Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Compositions and methods for conferring hydroxyphenyl pyruvate
dioxygenase (HPPD) herbicide resistance or tolerance to plants are
provided. Compositions include amino acid sequences, and variants and
fragments thereof, for mutant HPPD polypeptides. Nucleic acids that
encode the mutant HPPD polypeptides are also provided. Methods for
conferring herbicide resistance or tolerance, particularly resistance or
tolerance to certain classes of herbicides that inhibit HPPD, in plants
are further provided. Methods are also provided for selectively
controlling weeds in a field at a crop locus and for the assay,
characterization, identification and selection of the mutant HPPDs of the
current invention that provide herbicide tolerance.

Claims:

1. (canceled)

2. (canceled)

3. A polynucleotide encoding a polypeptide having at least 759 sequence
identity to SEQ ID NO:27, wherein said polypeptide has HPPD enzymatic
activity and comprises the amino acid sequence GFGKGNFSE (SEQ ID NO:70),
wherein the second G is replaced with any other amino acid.

4. (canceled)

5. (canceled)

6. The polynucleotide of claim 3, wherein the nucleotide sequence is
optimized for expression in a plant.

7. An expression cassette comprising the polynucleotide of claim 3
operably linked to a promoter that drives expression in a plant or plant
cell.

8. The expression cassette of claim 7 further comprising an operably
linked polynucleotide sequence encoding a polypeptide that confers a
desirable trait.

9. The expression cassette of claim 8, wherein said desirable trait is
resistance or tolerance to an herbicide.

10. The expression cassette of claim 9, wherein said desirable trait is
resistance or tolerance to an HPPD inhibitor, glyphosate, or glufosinate.

11. The expression cassette of claim 10, wherein said polypeptide that
confers a desirable trait is a cytochrome P450 or variant thereof.

12. The expression cassette of claim 10, wherein said polypeptide that
confers a desirable trait is an EPSPS
(5-enol-pyrovyl-shikimate-3-phosphate-synthase).

13. The expression cassette of claim 10, wherein said polypeptide that
confers a desirable trait is a phosphinothricin acetyl transferase.

14. A vector comprising an expression cassette according to claim 7.

15. The vector of claim 14, wherein said vector comprises a
polynucleotide comprising the sequence set forth in SEQ ID NO:33, 34, 35,
or 36.

16. A method for conferring resistance or tolerance to an HPPD inhibitor
in a plant, said method comprising introducing into said plant at least
one expression cassette according to claim 7.

17. A transformed plant cell comprising at least one expression cassette
according to claim 7.

21. A method of controlling weeds at a locus, said method comprising
applying to said locus a weed-controlling amount of one or more HPPD
inhibitors, wherein said locus comprises a plant according to claim 20.

22. The method of claim 21, wherein said HPPD inhibitor is selected from
the group consisting of: a) a compound of formula (Ia) ##STR00022##
wherein R1 and R2 are hydrogen or together form an ethylene
bridge; R3 is hydroxy or phenylthio-; R4 is halogen, nitro,
C1-C4 alkyl, C1-C4 alkoxy-C1-C4 alkyl-,
C1-C4 alkoxy-C1-C4 alkoxy-C1-C4 alkyl-; X
is methine, nitrogen, or C--R5 wherein R5 is hydrogen,
C1-C4 alkoxy, C1-C4 haloalkoxy-C1-C4
alkyl-, or a group ##STR00023## and R6 is C1-C4
alkylsulfonyl- or C1-C4 haloalkyl; b) a compound of formula
(Ib) ##STR00024## R1 and R2 are independently C1-C4
alkyl; and the free acids thereof; c) a compound of formula (Ic)
##STR00025## wherein R1 is hydroxy,
phenylcarbonyl-C1-C4alkoxy- or phenylcarbonyl-C1-C4
alkoxy- wherein the phenyl moiety is substituted in para-position by
halogen or C1-C4 alkyl, or phenylsulfonyloxy- or
phenylsulfonyloxy- wherein the phenyl moiety is substituted in
para-position by halogen or C1-C4 alkyl; R2 is
C1-C4 alkyl; R3 is hydrogen or C1-C4 alkyl;
R4 and R6 are independently halogen, C1-C4 alkyl,
C1-C4 haloalkyl, or C1-C4 alkylsulfonyl-; and R5
is hydrogen, C1-C4alkyl, C1-C4
alkoxy-C1-C4alkoxy-, or a group ##STR00026## d) a compound of
formula (Id) ##STR00027## wherein R1 is hydroxy; R2 is
C1-C4 alkyl; R3 is hydrogen; and R4, R5 and
R6 are independently C1-C4alkyl; e) a compound of formula
(Ie) ##STR00028## wherein R1 is cyclopropyl; R2 and R4
are independently halogen, C1-C4haloalkyl, or
C1-C4alkylsulfonyl-; and R3 is hydrogen; f) a compound of
formula (If) ##STR00029## wherein R1 is cyclopropyl; R2 and
R4 are independently halogen, C1-C4haloalkyl, or
C1-C4alkylsulfonyl-; and R3 is hydrogen; and g) a compound
of formula (Ig) or Formula (Ih) ##STR00030## wherein: -- R2 is
selected from the group consisting of C1-C3alkyl,
C1-C3-haloalkyl, C1-C3alkoxy-C1-C3alkyl and
C1-C3alkoxy-C2-C3alkoxy-C1-C3-alkyl;
R5 is hydrogen or methyl; R6 is selected from the group
consisting of hydrogen, fluorine, chlorine, hydroxyl and methyl; R7
is selected from the group consisting of hydrogen, halogen, hydroxyl,
sulfhydryl, C1-C6alkyl, C3-C6cycloalkyl,
C1-C6haloalkyl, C2-C6haloalkenyl, C2-C6
alkenyl, C3-C6alkynyl, C1-C6alkoxy,
C4-C7cycloalkoxy, C1-C6haloalkoxy,
C1-C6alkylthio, C1-C6alkylsulfinyl,
C1-C6alkylsulfonyl, C1-C6haloalkylthio, amino,
C1-C6alkylamino, C2-C6dialkylamino,
C2-C6dialkylaminosulfonyl, C1-C6alkylaminosulfonyl,
C1-C6alkoxy-C1-C6alkyl,
C1-C6alkoxy-C2-C6alkoxy,
C1-C6alkoxy-C2-C6 alkoxy-C1-C6-alkyl,
C3-C6alkenyl-C2-C6alkoxy,
C3-C6alkynyl-C1-C6alkoxy
C1-C6alkoxycarbonyl, C1-C5alkylcarbonyl,
C1-C4alkylenyl-S(O)p-R', C1-C4alkylenyl-CO2--R',
C1-C4alkylenyl-(CO)N--R'R', phenyl, phenylthio, phenylsulfinyl,
phenylsulfonyl, phenoxy, pyrrolidinyl, piperidinyl, morpholinyl and 5 or
6-membered heteroaryl or heteroaryloxy, the heteroaryl containing one to
three heteroatoms, each independently selected from the group consisting
of oxygen, nitrogen and sulphur, wherein the phenyl or heteroaryl
component may be optionally substituted by a substituent selected from
the group consisting of C1-C3alkyl, C1-C3haloalkyl,
C1-C3 alkoxy, C1-C3haloalkoxy, halo, cyano, and
nitro; X=O or S; n=0 or 1; m=0 or 1 with the proviso that if m=1 then n=0
and if n=1 then m=0; p=0, 1, or 2; R' is independently selected from the
group consisting of hydrogen and C1-C6alkyl; R8 is
selected from the group consisting of hydrogen, C1-C6alkyl,
C1-C6haloalkyl,
C1-C6alkylcarbonyl-C1-C3alkyl,
C3-C6cycloalkylalkeneyl for example cyclohexylmethylenyl,
C3-C6alkynylalkyleneyl for example propargyl,
C2-C5alkenylalkylenyl for example allyl, C1-C6alkoxy
C1-C6alkyl, cyano-C1-C6-alkyl,
arylcarbonyl-C1-C3-alkyl (wherein the aryl may be optionally
substituted with a substituent selected from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), aryl-C1-C6alkyl (wherein the aryl may be optionally
substituted with a substituent selected from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), C1-C6alkoxy C1-C6alkoxy
C1-C6alkyl and a 5 or 6-membered
heteroaryl-C1-C3-alkyl or heterocyclyl-C1-C3-alkyl,
the heteroaryl or heterocyclyl containing one to three heteroatoms, each
independently selected from the group consisting of oxygen, nitrogen and
sulphur, wherein the heterocyclyl or heteroaryl component may be
optionally substituted by a substituent selected from the group
consisting of halo, C1-C3alkyl, C1-C3haloalkyl, and
C1-C3 alkoxy; Q is selected from the group consisting of:
##STR00031## wherein A1 is selected from the group consisting of O,
C(O), S, SO, SO2 and (CReRf)q; q=0, 1 or 2; Ra,
Rb, Rc, Rd, Re and Rf are each independently
selected from the group consisting of C1-C4 alkyl which may be
mono-, di- or tri-substituted by substituents selected from the group
consisting of C1-C4 alkoxy, halogen, hydroxy, cyano,
hydroxycarbonyl, C1-C4 alkoxycarbonyl,
C1-C4alkylthio, C1-C4alkylsulfinyl,
C1-C4alkylsulfonyl, C1-C4 alkylcarbonyl, phenyl and
heteroaryl, it being possible for the phenyl and heteroaryl groups in
turn to be mono-, di- or tri-substituted by substituents selected from
the group consisting of C1-C4alkoxy, halogen, hydroxy, cyano,
hydroxycarbonyl, C1-C4alkoxycarbonyl,
C1-C4alkylsulfonyl and C1-C4haloalkyl, the
substituents on the nitrogen in the heterocyclic ring being other than
halogen; or Ra, Rb, Rc, Rd, Re and Rf are
each independently selected from the group consisting of hydrogen,
C1-C4alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylthio,
C1-C4alkylsulfinyl, C1-C4alkylsulfonyl,
C1-C4alkylcarbonyl, phenyl or heteroaryl, it being possible for
the phenyl and heteroaryl groups in turn to be mono-, di- or
tri-substituted by substituents selected from the group consisting of
C1-C4 alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylsulfonyl and
C1-C4haloalkyl, the substituents on the nitrogen in the
heterocyclic ring being other than halogen; or Ra and Rb
together form a 3- to 5-membered carbocyclic ring which may be
substituted by C1-C4alkyl and may be interrupted by oxygen,
sulfur, S(O), SO2, OC(O), NRg or by C(O); or Ra and
Rc together form a C1-C3alkylene chain which may be
interrupted by oxygen, sulfur, SO, SO2, OC(O), NRh or by C(O);
it being possible for that C1-C3alkylene chain in turn to be
substituted by C1-C4alkyl; Rg and Rh are each
independently of the other C1-C4alkyl, C1-C4
haloalkyl, C1-C4alkylsulfonyl, C1-C4alkylcarbonyl or
C1-C4alkoxycarbonyl; Ri is C1-C4alkyl; R3
is selected from the group consisting of C1-C6alkyl, optionally
substituted with halogen and/or C1-C3alkoxy; and
C3-C6 cycloalkyl optionally substituted with halogen and/or
C1-C3alkoxy; R9 is selected from the group consisting of
cyclopropyl, CF3 and i-Pr; R10 is selected from the group
consisting of hydrogen, I, Br, SR11, S(O)R11,
S(O)2R11; and R11 is C1-4 alkyl.

23. The method of claim 22, wherein said HPPD inhibitor is mesotrione.

24. The polynucleotide of claim 3, wherein the G is replaced with I in
the encoded polypeptide.

25. The polynucleotide of claim 3, wherein the G is replaced with A in
the encoded polypeptide.

26. The polynucleotide of claim 3, wherein the G is replaced with A in
the encoded polypeptide.

Description:

RELATED APPLICATIONS

[0001] Priority is claimed to U.S. Provisional Application No. 61/224,661,
filed Jul. 10, 2009, and to U.S. Provisional Application No. 61/146,513,
filed Jan. 22, 2009, which are incorporated herein by reference in their
entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to mutant hydroxyphenyl pyruvate
dioxygenase (HPPD) polypeptides that confer herbicide resistance or
tolerance to plants and the nucleic acid sequences that encode them.
Methods of the invention relate to the production and use of plants that
express these mutant HPPD polypeptides and that are resistant to HPPD
herbicides.

BACKGROUND OF THE INVENTION

[0003] The hydroxyphenylpyruvate dioxygenases (HPPDs) are enzymes that
catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is
transformed into homogentisate. This reaction takes place in the presence
of enzyme-bound iron (Fe2+) and oxygen. Herbicides that act by
inhibiting HPPD are well known, and include isoxazoles, diketonitriles,
triketones, and pyrazolinates (Hawkes "Hydroxyphenylpyruvate Dioxygenase
(HPPD)--The Herbicide Target." In Modern Crop Protection Compounds. Eds.
Kramer and Schirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp.
211-220). Inhibition of HPPD blocks the biosynthesis of plastoquinone
(PQ) from tyrosine. PQ is an essential cofactor in the biosynthesis of
carotenoid pigments which are essential for photoprotection of the
photosynthetic centres. HPPD-inhibiting herbicides are phloem-mobile
bleachers which cause the light-exposed new meristems and leaves to
emerge white. In the absence of carotenoids, chlorophyll is
photo-destroyed and becomes itself an agent of photo-destruction via the
photo-generation of singlet oxygen.

[0004] Methods are also known for providing plants that are tolerant to
HPPD herbicides and have included: 1) overexpressing the HPPD enzyme so
as to produce quantities of HPPD enzyme in the plant that are sufficient
in relation to a given herbicide so as to have enough of the functional
enzyme available despite the presence of its inhibitor; and 2) mutating
the target HPPD enzyme into a functional HPPD that is less sensitive to
herbicides. With respect to mutant HPPDs, while a given mutant HPPD
enzyme may provide a useful level of tolerance to some HPPD-inhibitor
herbicides, the same mutant HPPD may be quite inadequate to provide
commercial levels of tolerance to a different, more desirable
HPPD-inhibitor herbicide (See, e.g., U.S. App. Pub. No. 2004/0058427; and
PCT App. Pub. Nos. WO 98/20144 and WO 02/46387; see also U.S. App. Pub.
No. 2005/0246800 relating to identification and labelling of soybean
varieties as being relatively HPPD tolerant). For example, HPPD-inhibitor
herbicides may differ in terms of the spectrum of weeds they control,
their manufacturing cost, and their environmental benefits.

[0005] Accordingly, new methods and compositions for conferring HPPD
herbicide tolerance upon various crops and crop varieties are needed.

SUMMARY OF THE INVENTION

[0006] Compositions and methods for conferring hydroxyphenyl pyruvate
dioxygenase (HPPD) herbicide resistance or tolerance to plants are
provided. The compositions include nucleotide and amino acid sequences
for mutant HPPD polypeptides. The polypeptides of the invention are
mutant HPPDs that have HPPD enzymatic activity and that confer resistance
or tolerance in plants to certain classes of herbicides that inhibit
HPPD. In one embodiment, the compositions of the invention comprise a
mutant HPPD polypeptide having at least 80% sequence identity to SEQ ID
NO:27, where the polypeptide has HPPD enzymatic activity, and where the
polypeptide contains one or more amino acid additions, substitutions, or
deletions selected from the group consisting of:

[0007] 1) R(K,A,R)SQI(Q,E)T (SEQ ID NO:28), wherein the first Q is
replaced with any other amino acid, particularly with A, G, M, T, S, C,
R, F and more particularly with P;

[0041] 35) IFTKPVGDR (SEQ ID NO:65), wherein P is replaced with any other
amino acid; such as N;

[0042] 36) RPTFFLEMI (SEQ ID NO:66), wherein F is replaced with any other
amino acid; such as L;

[0043] 37) FLEMIQRIG (SEQ ID NO:67), wherein I is replaced with any other
amino acid; such as V or C;

[0044] 38) GGCGGFGKG (SEQ ID NO:68), wherein the fourth G is replaced with
any other amino acid; such as A, S, or T;

[0045] 39) GGFGKGNFS (SEQ ID NO:69), wherein K is replaced with any other
amino acid; such as L, A, E, or V;

[0046] 40) GFGKGNFSE (SEQ ID NO:70), wherein G is replaced with any other
amino acid; such as I;

[0047] 41) FGKGNFSEL (SEQ ID NO:71), wherein N is replaced with any other
amino acid; such as I;

[0048] 42) KGNFSELFK (SEQ ID NO:72), wherein S is replaced with any other
amino acid; such as N, G, K, or Q;

[0049] 43) GNFSELFKS (SEQ ID NO:73), wherein E is replaced with any other
amino acid; such as Q;

[0050] 44) ELFKSIEDY (SEQ ID NO:74), wherein S is replaced with any other
amino acid; such as A;

[0051] 45) LFKSIEDYE (SEQ ID NO:75), wherein I is replaced with any other
amino acid; such as L or F;

[0052] 46) HVVGNVPEM (SEQ ID NO:40), wherein N is replaced with any other
amino acid, particularly a C, and the amino acid sequence ELGVLVDRD (SEQ
ID NO:76), wherein the second L is replaced with any other amino acid,
particularly an M;

[0053] 47) LNSVVLANN (SEQ ID NO:47), wherein the second V is replaced with
any other amino acid, particularly an I, and the amino acid sequence
ELGVLVDRD (SEQ ID NO:76), wherein the second L is replaced with any other
amino acid, particularly an M;

[0054] 48) VLLPLNEPV (SEQ ID NO:50), wherein the third L is replaced with
any other amino acid, particularly an M, and the amino acid sequence
VLLQIFTKP (SEQ ID NO:62), wherein I is replaced with any other amino
acid, particularly a V;

[0055] 49) GGCGGFGKG (SEQ ID NO:68), wherein the fourth G is replaced with
any other amino acid, particularly a T, and the amino acid sequence
ELGVLVDRD (SEQ ID NO:76), wherein the second L is replaced with any other
amino acid, particularly an M;

[0056] 50) FHEFAEFTAED (SEQ ID NO:76), wherein the first A, the second E,
and the second F are replaced with any other amino acid, particularly
where the A is replaced with an S or a W, the E is replaced with a T,
and/or the F is replaced with an A or a V;

[0057] 51) HGTKRRSQIQ (SEQ ID NO:77), wherein the first R is replaced with
any other amino acid, particularly with a K, and the second R is deleted;

[0058] 52) GTKRRSQIQ (SEQ ID NO:78), wherein the second R is deleted;

[0059] 53) FMAPPQAKY (SEQ ID NO:59), wherein the second P is deleted;

[0060] 54) GNFSELFKS (SEQ ID NO:73), wherein the E is deleted;

[0061] 55) GVRRIAGDV (SEQ ID NO:61), wherein the I is deleted;

[0062] 56) DQGVLLQIFTKP (SEQ ID NO:79), wherein the first L and the I are
replaced with any other amino acid, particularly where the A is replaced
with an M and/or the I is replaced with an L;

[0063] 57) GKGNFSELFK (SEQ ID NO:80), wherein the F and the S are replaced
with any other amino acid, particularly where the F is replaced with a G
and/or the S is replaced with an A;

[0064] 58) KGNFSELFKS (SEQ ID NO:56), wherein the first S and the E are
replaced with any other amino acid, particularly where the S is replaced
with an N, G, or K and/or the E is replaced with an S or an A;

[0067] 61) ESGLN(S,G) (SEQ ID NO:31), wherein the first G is replaced with
any other amino acid, particularly with R, S, or A; and

[0068] 62) VLLPLNEPV (SEQ ID NO:50), wherein the second L is replaced with
any other amino acid, such as M, F, or V.

[0069] In another embodiment, the compositions of the invention comprise a
mutant HPPD polypeptide having at least 80% sequence identity to SEQ ID
NO:14 or to SEQ ID NO:27, where the polypeptide has HPPD enzymatic
activity, and where the polypeptide contains one or more amino acid
substitutions selected from the group consisting of:

[0083] 14) ESGLN(S,G) (SEQ ID NO:31), wherein the first G is replaced with
any other amino acid, particularly with R, S, or A; and

[0084] 15) VLLPLNEPV (SEQ ID NO:50), wherein the second L is replaced with
any other amino acid, such as M, F, or V.

[0085] Exemplary mutant HPPD polypeptides according to the invention
correspond to the amino acid sequences set forth in SEQ ID NOS:14-26, and
variants and fragments thereof. Nucleic acid molecules comprising
polynucleotide sequences that encode the mutant HPPD polypeptides of the
invention are further provided, e.g., SEQ ID NOS:1-13. Compositions also
include expression cassettes comprising a promoter operably linked to a
nucleotide sequence that encodes a mutant HPPD polypeptide of the
invention, alone or in combination with one or more additional nucleic
acid molecules encoding polypeptides that confer desirable traits.
Transformed plants, plant cells, and seeds comprising an expression
cassette of the invention are further provided.

[0086] The compositions of the invention are useful in methods directed to
conferring herbicide resistance or tolerance to plants, particularly
resistance or tolerance to certain classes of herbicides that inhibit
HPPD. In particular embodiments, the methods comprise introducing into a
plant at least one expression cassette comprising a promoter operably
linked to a nucleotide sequence that encodes a mutant HPPD polypeptide of
the invention. As a result, the mutant HPPD polypeptide is expressed in
the plant, and the mutant HPPD is less sensitive to HPPD-inhibiting
herbicides, thereby leading to resistance or tolerance to HPPD-inhibiting
herbicides.

[0087] Methods of the present invention also comprise selectively
controlling weeds in a field at a crop locus. In one embodiment, such
methods involve over-the-top pre- or postemergence application of
weed-controlling amounts of HPPD herbicides in a field at a crop locus
that contains plants expressing the mutant HPPD polypeptides of the
invention. In other embodiments, methods are also provided for the assay,
characterization, identification, and selection of the mutant HPPDs of
the current invention.

[0089] FIGS. 2A-2B show on rate (FIG. 2A) and off rate (FIG. 2B)
determinations for a complex of structure B with the HPPD polypeptide
corresponding to the amino acid sequence set forth in SEQ ID NO:14.

[0090]FIG. 3 shows an off rate determination for a complex of structure D
with the HPPD polypeptide corresponding to the amino acid sequence set
forth in SEQ ID NO:14.

[0091] FIGS. 4A-4C show off rate determinations at ice temperature for
complexes of structure B with the HPPD polypeptides corresponding to the
amino acid sequences set forth in SEQ ID NO:14 (FIG. 4A), 24 (FIG. 4B),
and 26 (FIG. 4C).

[0092] FIG. 5 shows mesotrione inhibition of pyomelanin formation by E.
coli BL21 expressing different variants of HPPD. Left bar=(error range
for n=3) average A 430 nm with zero mesotrione present in the medium and
right bar=(n=3) average A 430 nm with 12.5 ppm present in the medium.
Control is pET24 empty vector where no HPPD is expressed.

[0098] The present invention provides compositions and methods directed to
conferring hydroxyphenyl pyruvate dioxygenase (HPPD) herbicide resistance
or tolerance to plants. Compositions include amino acid sequences for
mutant HPPD polypeptides having HPPD enzymatic activity, and variants and
fragments thereof. Nucleic acids that encode the mutant HPPD polypeptides
of the invention are also provided. Methods for conferring herbicide
resistance or tolerance to plants, particularly resistance or tolerance
to certain classes of herbicides that inhibit HPPD, are further provided.
Methods are also provided for selectively controlling weeds in a field at
a crop locus and for the assay, characterization, identification and
selection of the mutant HPPDs of the current invention that provide
herbicide tolerance.

[0100] "HPPD herbicides" are herbicides that are bleachers and whose
primary site of action is HPPD. Many are well known and described
elsewhere herein and in the literature (Hawkes "Hydroxyphenylpyruvate
Dioxygenase (HPPD)--The Herbicide Target." In Modern Crop Protection
Compounds. Eds. Kramer and Schirmer. Weinheim, Germany: Wiley-VCH, 2007.
Ch. 4.2, pp. 211-220; Edmunds "Hydroxyphenylpyruvate dioxygenase (HPPD)
Inhibitors: Triketones." In Modern Crop Protection Compounds. Eds. Kramer
and Schirmer. Weinheim, Germany: Wiley-VCH, 2007. Ch. 4.2, pp. 221-242).
As used herein, the term "HPPD herbicides" refers to herbicides that act
either directly or indirectly to inhibit HPPD, where the herbicides are
bleachers, and where inhibition of HPPD is at least part of the
herbicide's mode of action on plants.

[0101] As used herein, plants which are substantially "tolerant" to a
herbicide exhibit, when treated with said herbicide, a dose/response
curve which is shifted to the right when compared with that exhibited by
similarly subjected non tolerant like plants. Such dose/response curves
have "dose" plotted on the x-axis and "percentage kill or damage",
"herbicidal effect" etc. plotted on the y-axis. Tolerant plants will
typically require at least twice as much herbicide as non tolerant like
plants in order to produce a given herbicidal effect. Plants which are
substantially "resistant" to the herbicide exhibit few, if any, necrotic,
lytic, chlorotic or other lesions or, at least, none that impact
significantly on yield, when subjected to the herbicide at concentrations
and rates which are typically employed by the agricultural community to
kill weeds in the field.

[0102] As used herein, "non-transgenic-like plants" are plants that are
similar or the same as transgenic plants but that do not contain a
transgene conferring herbicide resistance.

[0103] As used herein, the term "confer" refers to providing a
characteristic or trait, such as herbicide tolerance or resistance and/or
other desirable traits to a plant.

[0104] As described elsewhere herein, the term "heterologous" means from
another source. In the context of DNA, "heterologous" refers to any
foreign "non-self" DNA including that from another plant of the same
species. For example, in the present application a soybean HPPD gene that
was transgenically expressed back into a soybean plant would still be
described as "heterologous" DNA.

[0105] The article "a" and "an" are used herein to refer to one or more
than one (i.e., to at least one) of the grammatical object of the
article. By way of example, "an element" means one or more element.
Throughout the specification the word "comprising," or variations such as
"comprises" or "comprising," will be understood to imply the inclusion of
a stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step, or
group of elements, integers or steps.

[0106] A variety of additional terms are defined or otherwise
characterized herein.

HPPD Sequences

[0107] The compositions of the invention include isolated or substantially
purified mutant HPPD polynucleotides and polypeptides as well as host
cells comprising mutant HPPD polynucleotides. Specifically, the present
invention provides mutant HPPD polypeptides that have HPPD enzymatic
activity and that confer resistance or tolerance in plants to certain
classes of herbicides that inhibit HPPD, and variants and fragments
thereof. Nucleic acids that encode the mutant HPPD polypeptides of the
invention are also provided.

[0108] Mutant HPPD polypeptides of the presenting invention have amino
acid changes at one or more positions relative to the starting wild type
sequence from which they are derived, and exhibit enhanced tolerance to
one or more HPPD inhibitor herbicides. HPPD enzymes that exhibit enhanced
tolerance to an HPPD herbicide may do so by virtue of exhibiting,
relative to the like unmutated starting enzyme:

[0114] e) as a result of changes in one or both of c) and d), an increased
value of the equilibrium constant, Ki (also called Kd), governing
dissociation of an enzyme: HPPD inhibitor herbicide complex. DNA
sequences encoding such improved mutated HPPDs are used in the provision
of HPPD plants, crops, plant cells and seeds of the current invention
that offer enhanced tolerance or resistance to one or more HPPD
herbicides as compared to like plants likewise expressing the unmutated
starting enzyme.

[0115] Increases in the value of koff are of particular value in improving
the ability of HPPD to confer resistance to a HPPD herbicide. As one
example, compounds B and C exhibit similar Kd values with respect to the
HPPD variant of SEQ ID NO:14 but differ in that the koff value for
compound B is about 10-fold greater as compared to the koff value for
compound C, and plants expressing SEQ ID NO:14 show superior resistance
to compound B than to compound C.

[0116] Site-directed mutations of genes encoding plant-derived HPPDs are
selected so as to encode amino acid changes selected from the list below
either singly or in combination. Genes encoding such mutant forms of
plant HPPDs are useful for making crop plants resistant to herbicides
that inhibit HPPD. Plant HPPD genes so modified are especially suitable
for use in transgenic plants in order to confer herbicide tolerance or
resistance upon crop plants.

[0117] Many HPPD sequences are known in the art and can be used to
generate mutant HPPD sequences by making the corresponding amino acid
substitutions, deletions, and additions described herein. The HPPD amino
acid sequence of Avena sativa is set forth in SEQ ID NO:27. A single
deletion variant of the Avena sativa HPPD is set forth in SEQ ID NO:14.
Thus, a known or suspected HPPD sequence can be aligned with, for
example, SEQ ID NO:14 or SEQ ID NO:27 using standard sequence alignment
tools, and the corresponding amino acid substitutions, deletions, and/or
additions described herein with respect to SEQ ID NO:14 or to SEQ ID
NO:27 can be made in the reference sequence.

[0118] In one embodiment, the compositions of the invention comprise a
mutant HPPD polypeptide having at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more sequence identity to SEQ ID NO:27 (the HPPD amino acid sequence
of Avena sativa) or where the HPPD amino acid sequence derives from a
plant, where the polypeptide has HPPD enzymatic activity, and where the
polypeptide contains one or more amino acid sequence additions,
substitutions, or deletions corresponding to the amino acid positions
listed in column 1 of Table 1, optionally in further combination with
known mutations (see e.g., WO2009/144079). In various embodiments, an
amino acid at one or more position(s) listed in column 1 of Table 1 is
replaced with any other amino acid. In another embodiment, the
polypeptide comprises one or more amino acid substitutions, additions, or
deletions corresponding to the amino acid substitutions or additions
listed in column 2 of Table 1. In yet another embodiment, the polypeptide
comprises one or more substitutions corresponding to a conservative
variant of the amino acids listed in column 2 of Table 1. For example,
the polypeptide may comprise a mutation corresponding to amino acid
position 217 of SEQ ID NO:14 (amino acid position 218 of SEQ ID NO:27),
wherein that amino acid is replaced with alanine or a conservative
substitution of alanine; or the polypeptide may comprise a mutation
corresponding to amino acid position 241 of SEQ ID NO:14 (amino acid
position 242 of SEQ ID NO:27), wherein that amino acid is replaced with
tryptophan or a conservative substitution of tryptophan; or the
polypeptide may comprise a mutation corresponding to amino acid position
408 of SEQ ID NO:14 (amino acid position 409 of SEQ ID NO:27), wherein
that amino acid is replaced with alanine or a conservative substitution
of alanine. In particular embodiments, the amino acid sequence of the
mutant HPPD polypeptide of the invention is selected from the group
consisting of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
and 26.

[0119] In another embodiment, the compositions of the invention comprise a
mutant HPPD polypeptide having at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more sequence identity to SEQ ID NO:27 (the HPPD amino acid sequence
of Avena sativa) or where the HPPD amino acid sequence derives from a
plant, where the polypeptide has HPPD enzymatic activity, and where the
polypeptide contains one or more amino acid sequence substitutions
corresponding to the amino acid positions listed in column 1 of Table 2,
optionally in further combination with known mutations (see e.g.,
WO2009/144079). In various embodiments, an amino acid at one or more
position(s) listed in column 1 of Table 2 is replaced with any other
amino acid. In another embodiment, the polypeptide comprises one or more
amino acid substitutions corresponding to the amino acid substitutions
listed in column 2 of Table 2. In yet another embodiment, the polypeptide
comprises one or more substitutions corresponding to a conservative
variant of the amino acids listed in column 2 of Table 2. For example,
the polypeptide may comprise a mutation corresponding to amino acid
position 217 of SEQ ID NO:14 (amino acid position 218 of SEQ ID NO:27),
wherein that amino acid is replaced with alanine or a conservative
substitution of alanine; or the polypeptide may comprise a mutation
corresponding to amino acid position 241 of SEQ ID NO:14 (amino acid
position 242 of SEQ ID NO:27), wherein that amino acid is replaced with
tryptophan or a conservative substitution of tryptophan; or the
polypeptide may comprise a mutation corresponding to amino acid position
408 of SEQ ID NO:14 (amino acid position 409 of SEQ ID NO:27), wherein
that amino acid is replaced with alanine or a conservative substitution
of alanine In particular embodiments, the amino acid sequence of the
mutant HPPD polypeptide of the invention is selected from the group
consisting of SEQ ID NO:14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
and 26.

[0120] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. The
terms apply to amino acid polymers in which one or more amino acid
residues is an artificial chemical analogue of a corresponding naturally
occurring amino acid, as well as to naturally occurring amino acid
polymers. Polypeptides of the invention can be produced either from a
nucleic acid disclosed herein, or by the use of standard molecular
biology techniques. For example, a truncated protein of the invention can
be produced by expression of a recombinant nucleic acid of the invention
in an appropriate host cell, or alternatively by a combination of ex vivo
procedures, such as protease digestion and purification.

[0121] Accordingly, the present invention also provides nucleic acid
molecules comprising polynucleotide sequences that encode mutant HPPD
polypeptides that have HPPD enzymatic activity and that confer resistance
or tolerance in plants to certain classes of herbicides that inhibit
HPPD, and variants and fragments thereof. In general, the invention
includes any polynucleotide sequence that encodes any of the mutant HPPD
polypeptides described herein, as well as any polynucleotide sequence
that encodes HPPD polypeptides having one or more conservative amino acid
substitutions relative to the mutant HHPD polypeptides described herein.
Conservative substitution tables providing functionally similar amino
acids are well known in the art. The following five groups each contain
amino acids that are conservative substitutions for one another:
Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine
(I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,
Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),
Asparagine (N), Glutamine (Q).

[0122] In one embodiment, the present invention provides a polynucleotide
sequence encoding an amino acid sequence having at least about 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:14 or to SEQ ID
NO:27 or where the HPPD amino acid sequence derives from a plant, where
the polypeptide has HPPD enzymatic activity, and where the polypeptide
contains one or more amino acid sequence additions, substitutions, or
deletions as described herein. In particular embodiments, the
polynucleotide sequence encodes a mutant HPPD polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID NO:14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In another embodiment, the
present invention provides a polynucleotide sequence selected from the
group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and
13.

[0123] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses known
analogues (e.g., peptide nucleic acids) having the essential nature of
natural nucleotides in that they hybridize to single-stranded nucleic
acids in a manner similar to naturally occurring nucleotides.

[0124] As used herein, the terms "encoding" or "encoded" when used in the
context of a specified nucleic acid mean that the nucleic acid comprises
the requisite information to direct translation of the nucleotide
sequence into a specified protein. The information by which a protein is
encoded is specified by the use of codons. A nucleic acid encoding a
protein may comprise non-translated sequences (e.g., introns) within
translated regions of the nucleic acid or may lack such intervening
non-translated sequences (e.g., as in cDNA).

[0125] The invention encompasses isolated or substantially purified
polynucleotide or protein compositions. An "isolated" or "purified"
polynucleotide or protein, or biologically active portion thereof, is
substantially or essentially free from components that normally accompany
or interact with the polynucleotide or protein as found in its naturally
occurring environment. Thus, an isolated or purified polynucleotide or
protein is substantially free of other cellular material, or culture
medium when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
Optimally, an "isolated" polynucleotide is free of sequences (optimally
protein encoding sequences) that naturally flank the polynucleotide
(i.e., sequences located at the 5' and 3' ends of the polynucleotide) in
the genomic DNA of the organism from which the polynucleotide is derived.
For example, in various embodiments, the isolated polynucleotide can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb
of nucleotide sequence that naturally flank the polynucleotide in genomic
DNA of the cell from which the polynucleotide is derived. A protein that
is substantially free of interfering enzyme activities and that is
capable of being characterized in respect of its catalytic, kinetic and
molecular properties includes quite crude preparations of protein (for
example recombinantly produced in cell extracts) having less than about
98%, 95% 90%, 80%, 70%, 60% or 50% (by dry weight) of contaminating
protein as well as preparations further purified by methods known in the
art to have 40%, 30%, 20%, 10%, 5%, or 1% (by dry weight) of
contaminating protein.

[0126] The proteins of the invention may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in the
art. For example, amino acid sequence variants and fragments of the
mutant HPPD proteins can be prepared by mutations in the DNA. Methods for
mutagenesis and polynucleotide alterations are well known in the art.
See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.
4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York) and the references cited
therein. Guidance as to appropriate amino acid substitutions that often
do not affect biological activity of the protein of interest may be found
in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and
Structure (Natl. Biomed. Res. Found, Washington, D.C.), herein
incorporated by reference. Conservative substitutions, such as exchanging
one amino acid with another having similar properties, may be optimal.

[0127] The polynucleotides of the invention can also be used to isolate
corresponding sequences from other organisms, particularly other plants.
In this manner, methods such as PCR, hybridization, and the like can be
used to identify such sequences based on their sequence homology to the
sequences set forth herein.

[0128] In a PCR approach, oligonucleotide primers can be designed for use
in PCR reactions to amplify corresponding DNA sequences from cDNA or
genomic DNA extracted from any plant of interest. Methods for designing
PCR primers and PCR cloning are generally known in the art. See, for
example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual
(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also
Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and
Applications (Academic Press, New York); Innis and Gelfand, eds. (1995)
PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.
(1999) PCR Methods Manual (Academic Press, New York).

[0129] In hybridization techniques, all or part of a known polynucleotide
is used as a probe that selectively hybridizes to other corresponding
polynucleotides present in a population of cloned genomic DNA fragments
or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen
organism. The hybridization probes may be genomic DNA fragments, cDNA
fragments, RNA fragments, or other oligonucleotides, and may be labeled
with a detectable group such as 32P, or any other detectable marker.
Methods for preparation of probes for hybridization and for construction
of cDNA and genomic libraries are generally known in the art and are
disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0130] By "hybridizing to" or "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under stringent conditions when that sequence is
present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially" refers to complementary hybridization between a probe
nucleic acid and a target nucleic acid and embraces minor mismatches that
can be accommodated by reducing the stringency of the hybridization media
to achieve the desired detection of the target nucleic acid sequence.

[0131] "Stringent hybridization conditions" and "stringent hybridization
wash conditions" in the context of nucleic acid hybridization experiments
such as Southern and Northern hybridizations are sequence dependent, and
are different under different environmental parameters. Longer sequences
hybridize specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993) Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with
Nucleic Acid Probes part I chapter 2 "Overview of principles of
hybridization and the strategy of nucleic acid probe assays" Elsevier,
New York. Generally, highly stringent hybridization and wash conditions
are selected to be about 5° C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength and
pH. Typically, under "stringent conditions" a probe will hybridize to its
target subsequence, but to no other sequences.

[0132] The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly matched
probe. Very stringent conditions are selected to be equal to the Tm
for a particular probe. An example of stringent hybridization conditions
for hybridization of complementary nucleic acids which have more than 100
complementary residues on a filter in a Southern or northern blot is 50%
formamide with 1 mg of heparin at 42° C., with the hybridization
being carried out overnight. An example of highly stringent wash
conditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An
example of stringent wash conditions is a 0.2×SSC wash at
65° C. for 15 minutes (see, Sambrook, infra, for a description of
SSC buffer). Often, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example medium
stringency wash for a duplex of, e.g., more than 100 nucleotides, is
1×SSC at 45° C. for 15 minutes. An example low stringency
wash for a duplex of, e.g., more than 100 nucleotides, is 4-6×SSC
at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50
nucleotides), stringent conditions typically involve salt concentrations
of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion
concentration (or other salts) at pH 7.0 to 8.3, and the temperature is
typically at least about 30° C. Stringent conditions can also be
achieved with the addition of destabilizing agents such as formamide. In
general, a signal to noise ratio of 2× (or higher) than that
observed for an unrelated probe in the particular hybridization assay
indicates detection of a specific hybridization. Nucleic acids that do
not hybridize to each other under stringent conditions are still
substantially identical if the proteins that they encode are
substantially identical. This occurs, e.g., when a copy of a nucleic acid
is created using the maximum codon degeneracy permitted by the genetic
code.

[0134] Fragments and variants of the disclosed nucleotide sequences and
proteins encoded thereby are also encompassed by the present invention.
"Fragment" is intended to mean a portion of the nucleotide sequence or a
portion of the amino acid sequence and hence protein encoded thereby.
Fragments of a nucleotide sequence may encode protein fragments that
retain the biological activity of the mutant HPPD protein and hence have
HPPD enzymatic activity. Alternatively, fragments of a nucleotide
sequence that are useful as hybridization probes or in mutagenesis and
shuffling reactions to generate yet further HPPD variants generally do
not encode fragment proteins retaining biological activity. Thus,
fragments of a nucleotide sequence may range from at least about 20
nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the
full-length nucleotide sequence encoding the polypeptides of the
invention.

[0135] A fragment of a nucleotide sequence that encodes a biologically
active portion of a mutant HPPD protein of the invention will encode at
least 15, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 180, 200,
250, 300, or 350 contiguous amino acids, or up to the total number of
amino acids present in a full-length mutant HPPD polypeptide of the
invention. Fragments of a nucleotide sequence that are useful as
hybridization probes or PCR primers generally need not encode a
biologically active portion of an HPPD protein.

[0136] As used herein, "full-length sequence" in reference to a specified
polynucleotide means having the entire nucleic acid sequence of a native
or mutated HPPD sequence. "Native sequence" is intended to mean an
endogenous sequence, i.e., a non-engineered sequence found in an
organism's genome.

[0137] Thus, a fragment of a nucleotide sequence of the invention may
encode a biologically active portion of a mutant HPPD polypeptide, or it
may be a fragment that can be used as a hybridization probe etc. or PCR
primer using methods disclosed below. A biologically active portion of a
mutant HPPD polypeptide can be prepared by isolating a portion of one of
the nucleotide sequences of the invention, expressing the encoded portion
of the mutant HPPD protein (e.g., by recombinant expression in vitro),
and assessing the activity of the encoded portion of the mutant HPPD
protein. Nucleic acid molecules that are fragments of a nucleotide
sequence of the invention comprise at least 15, 20, 50, 75, 100, 150,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300
contiguous nucleotides, or up to the number of nucleotides present in a
full-length nucleotide sequence disclosed herein.

[0138] "Variants" is intended to mean substantially similar sequences. For
polynucleotides, a variant comprises a deletion and/or addition of one or
more nucleotides at one or more internal sites within the reference
polynucleotide and/or a substitution of one or more nucleotides at one or
more sites in the mutant HPPD polynucleotide. As used herein, a
"reference" polynucleotide or polypeptide comprises a mutant HPPD
nucleotide sequence or amino acid sequence, respectively. As used herein,
a "native" polynucleotide or polypeptide comprises a naturally occurring
nucleotide sequence or amino acid sequence, respectively. One of skill in
the art will recognize that variants of the nucleic acids of the
invention will be constructed such that the open reading frame is
maintained. For polynucleotides, conservative variants include those
sequences that, because of the degeneracy of the genetic code, encode the
amino acid sequence of one of the mutant HPPD polypeptides of the
invention. Naturally occurring allelic variants such as these can be
identified with the use of well-known molecular biology techniques, as,
for example, with polymerase chain reaction (PCR) and hybridization
techniques as outlined below. Variant polynucleotides also include
synthetically derived polynucleotide, such as those generated, for
example, by using site-directed mutagenesis but which still encode a
mutant HPPD protein of the invention. Generally, variants of a particular
polynucleotide of the invention will have at least about 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more sequence identity to that particular polynucleotide
as determined by sequence alignment programs and parameters described
elsewhere herein.

[0139] Variants of a particular polynucleotide of the invention (i.e., the
reference polynucleotide) can also be evaluated by comparison of the
percent sequence identity between the polypeptide encoded by a variant
polynucleotide and the polypeptide encoded by the reference
polynucleotide. Thus, for example, a polynucleotide that encodes a
polypeptide with a given percent sequence identity to the polypeptides of
SEQ ID NOS: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, are
disclosed. Percent sequence identity between any two polypeptides can be
calculated using sequence alignment programs and parameters described
elsewhere herein. Where any given pair of polynucleotides of the
invention is evaluated by comparison of the percent sequence identity
shared by the two polypeptides they encode, the percent sequence identity
between the two encoded polypeptides is at least about 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more sequence identity across the entirety of the HPPD
sequences described herein, i.e., when compared to the full length HPPD
sequences described herein.

[0140] "Variant" protein is intended to mean a protein derived from the
reference protein by deletion or addition of one or more amino acids at
one or more internal sites in the mutant HPPD protein and/or substitution
of one or more amino acids at one or more sites in the mutant HPPD
protein. Variant proteins encompassed by the present invention are
biologically active, that is they continue to possess the desired
biological activity of the mutant HPPD protein, that is, HPPD enzymatic
activity and/or herbicide tolerance as described herein. Such variants
may result from, for example, genetic polymorphism or from human
manipulation. Biologically active variants of a mutant HPPD protein of
the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity across the entirety of the amino acid sequence for the
mutant HPPD protein as determined by sequence alignment programs and
parameters described elsewhere herein. A biologically active variant of a
protein of the invention may differ from that protein by as few as 1-15
amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as
4, 3, 2, or even 1 amino acid residue.

[0141] Methods of alignment of sequences for comparison are well known in
the art and can be accomplished using mathematical algorithms such as the
algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment
algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global
alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48:443-453; and the algorithm of Karlin and Altschul (1990) Proc. Natl.
Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc.
Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of these
mathematical algorithms can be utilized for comparison of sequences to
determine sequence identity. Such implementations include, but are not
limited to: CLUSTAL in the PC/Gene program (available from
Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0)
and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton
Road, San Diego, Calif., USA).

Gene Stacking

[0142] In certain embodiments the polynucleotides of the invention
encoding mutant HPPD polypeptides or variants thereof that retain HPPD
enzymatic activity (e.g., a polynucleotide sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NO:14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, and 26) can be stacked with any
combination of polynucleotide sequences of interest in order to create
plants with a desired trait. A trait, as used herein, refers to the
phenotype derived from a particular sequence or groups of sequences. For
example, the polynucleotides encoding a mutant HPPD polypeptide or
variant thereof that retains HPPD enzymatic activity may be stacked with
any other polynucleotides encoding polypeptides that confer a desirable
trait, including but not limited to resistance to diseases, insects, and
herbicides, tolerance to heat and drought, reduced time to crop maturity,
improved industrial processing, such as for the conversion of starch or
biomass to fermentable sugars, and improved agronomic quality, such as
high oil content and high protein content.

[0144] Thus, in one embodiment, the polynucleotides encoding a mutant HPPD
polypeptide or variant thereof that retains HPPD enzymatic activity are
stacked with one or more polynucleotides encoding polypeptides that
confer resistance or tolerance to an herbicide. In one embodiment, the
desirable trait is resistance or tolerance to an HPPD inhibitor. In
another embodiment, the desirable trait is resistance or tolerance to
glyphosate. In another embodiment, the desirable trait is resistance or
tolerance to glufosinate.

[0145] These stacked combinations can be created by any method including,
but not limited to, cross-breeding plants by any conventional or TopCross
methodology, or genetic transformation. If the sequences are stacked by
genetically transforming the plants, the polynucleotide sequences of
interest can be combined at any time and in any order. For example, a
transgenic plant comprising one or more desired traits can be used as the
target to introduce further traits by subsequent transformation. The
traits can be introduced simultaneously in a co-transformation protocol
with the polynucleotides of interest provided by any combination of
transformation cassettes. For example, if two sequences will be
introduced, the two sequences can be contained in separate transformation
cassettes (trans) or contained on the same transformation cassette (cis).
Expression of the sequences can be driven by the same promoter or by
different promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of the
polynucleotide of interest. This may be combined with any combination of
other suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant. It is further recognized that
polynucleotide sequences can be stacked at a desired genomic location
using a site-specific recombination system. See, for example, WO99/25821,
WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are
herein incorporated by reference.

Plant Expression Cassettes

[0146] The compositions of the invention may additionally contain nucleic
acid sequences for transformation and expression in a plant of interest.
The nucleic acid sequences may be present in DNA constructs or expression
cassettes. "Expression cassette" as used herein means a nucleic acid
molecule capable of directing expression of a particular nucleotide
sequence in an appropriate host cell, comprising a promoter operatively
linked to the nucleotide sequence of interest (i.e., a polynucleotide
encoding a mutant HPPD polypeptide or variant thereof that retains HPPD
enzymatic activity, alone or in combination with one or more additional
nucleic acid molecules encoding polypeptides that confer desirable
traits) which is operatively linked to termination signals. It also
typically comprises sequences required for proper translation of the
nucleotide sequence. The coding region usually codes for a protein of
interest but may also code for a functional RNA of interest, for example
antisense RNA or a nontranslated RNA, in the sense or antisense
direction. The expression cassette comprising the nucleotide sequence of
interest may be chimeric, meaning that at least one of its components is
heterologous with respect to at least one of its other components. The
expression cassette may also be one that is naturally occurring but has
been obtained in a recombinant form useful for heterologous expression.
Typically, however, the expression cassette is heterologous with respect
to the host, i.e., the particular DNA sequence of the expression cassette
does not occur naturally in the host cell and must have been introduced
into the host cell or an ancestor of the host cell by a transformation
event. The expression of the nucleotide sequence in the expression
cassette may be under the control of a constitutive promoter or of an
inducible promoter that initiates transcription only when the host cell
is exposed to some particular external stimulus. Additionally, the
promoter can also be specific to a particular tissue or organ or stage of
development.

[0147] The present invention encompasses the transformation of plants with
expression cassettes capable of expressing a polynucleotide of interest,
i.e., a polynucleotide encoding a mutant HPPD polypeptide or variant
thereof that retains HPPD enzymatic activity, alone or in combination
with one or more additional nucleic acid molecules encoding polypeptides
that confer desirable traits. The expression cassette will include in the
5'-3' direction of transcription, a transcriptional and translational
initiation region (i.e., a promoter) and a polynucleotide open reading
frame. The expression cassette may optionally comprise a transcriptional
and translational termination region (i.e. termination region) functional
in plants. In some embodiments, the expression cassette comprises a
selectable marker gene to allow for selection for stable transformants.
Expression constructs of the invention may also comprise a leader
sequence and/or a sequence allowing for inducible expression of the
polynucleotide of interest. See, Guo et al. (2003) Plant J. 34:383-92 and
Chen et al. (2003) Plant J. 36:731-40 for examples of sequences allowing
for inducible expression.

[0148] The regulatory sequences of the expression construct are operably
linked to the polynucleotide of interest. By "operably linked" is
intended a functional linkage between a promoter and a second sequence
wherein the promoter sequence initiates and mediates transcription of the
DNA sequence corresponding to the second sequence. Generally, operably
linked means that the nucleotide sequences being linked are contiguous.

[0149] Any promoter capable of driving expression in the plant of interest
may be used in the practice of the invention. The promoter may be native
or analogous or foreign or heterologous to the plant host. The terms
"heterologous" and "exogenous" when used herein to refer to a nucleic
acid sequence (e.g. a DNA or RNA sequence) or a gene, refer to a sequence
that originates from a source foreign to the particular host cell or, if
from the same source, is modified from its original form. Thus, a
heterologous gene in a host cell includes a gene that is endogenous to
the particular host cell but has been modified through, for example, the
use of DNA shuffling. The terms also include non-naturally occurring
multiple copies of a naturally occurring DNA sequence. Thus, the terms
refer to a DNA segment that is foreign or heterologous to the cell, or
homologous to the cell but in a position within the host cell nucleic
acid in which the element is not ordinarily found. Exogenous DNA segments
are expressed to yield exogenous polypeptides.

[0150] A "homologous" nucleic acid (e.g. DNA) sequence is a nucleic acid
(e.g. DNA or RNA) sequence naturally associated with a host cell into
which it is introduced.

[0151] The choice of promoters to be included depends upon several
factors, including, but not limited to, efficiency, selectability,
inducibility, desired expression level, and cell- or tissue-preferential
expression. It is a routine matter for one of skill in the art to
modulate the expression of a sequence by appropriately selecting and
positioning promoters and other regulatory regions relative to that
sequence. The promoters that are used for expression of the transgene(s)
can be a strong plant promoter, a viral promoter, or a chimeric promoters
composed of elements such as: TATA box from any gene (or synthetic, based
on analysis of plant gene TATA boxes), optionally fused to the region 5'
to the TATA box of plant promoters (which direct tissue and temporally
appropriate gene expression), optionally fused to 1 or more enhancers
(such as the 35S enhancer, FMV enhancer, CMP enhancer, RUBISCO SMALL
SUBUNIT enhancer, PLASTOCYANIN enhancer).

[0153] Appropriate plant or chimeric promoters are useful for applications
such as expression of transgenes in certain tissues, while minimizing
expression in other tissues, such as seeds, or reproductive tissues.
Exemplary cell type- or tissue-preferential promoters drive expression
preferentially in the target tissue, but may also lead to some expression
in other cell types or tissues as well. Methods for identifying and
characterizing promoter regions in plant genomic DNA include, for
example, those described in the following references: Jordano, et al.,
Plant Cell, 1:855-866 (1989); Bustos, et al., Plant Cell, 1:839-854
(1989); Green, et al., EMBO J. 7, 4035-4044 (1988); Meier, et al., Plant
Cell, 3, 309-316 (1991); and Zhang, et al., Plant Physiology 110:
1069-1079 (1996).

[0154] In other embodiments of the present invention, inducible promoters
may be desired. Inducible promoters drive transcription in response to
external stimuli such as chemical agents or environmental stimuli. For
example, inducible promoters can confer transcription in response to
hormones such as giberellic acid or ethylene, or in response to light or
drought.

[0155] A variety of transcriptional terminators are available for use in
expression cassettes. These are responsible for the termination of
transcription beyond the transgene and correct mRNA polyadenylation. The
termination region may be native with the transcriptional initiation
region, may be native with the operably linked DNA sequence of interest,
may be native with the plant host, or may be derived from another source
(i.e., foreign or heterologous to the promoter, the DNA sequence of
interest, the plant host, or any combination thereof). Appropriate
transcriptional terminators are those that are known to function in
plants and include the CAMV 35S terminator, the tml terminator, the
nopaline synthase terminator and the pea rbcs E9 terminator. These can be
used in both monocotyledons and dicotyledons. In addition, a gene's
native transcription terminator may be used.

[0156] Generally, the expression cassette will comprise a selectable
marker gene for the selection of transformed cells. Selectable marker
genes are utilized for the selection of transformed cells or tissues.

[0157] Numerous sequences have been found to enhance gene expression from
within the transcriptional unit and these sequences can be used in
conjunction with the genes of this invention to increase their expression
in transgenic plants.

[0158] Various intron sequences have been shown to enhance expression,
particularly in monocotyledonous cells. For example, the introns of the
maize Adh1 gene have been found to significantly enhance the expression
of the wild-type gene under its cognate promoter when introduced into
maize cells. Intron 1 was found to be particularly effective and enhanced
expression in fusion constructs with the chloramphenicol
acetyltransferase gene (Callis et al., Genes Develop. 1:1183-1200
(1987)). In the same experimental system, the intron from the maize
bronze 1 gene had a similar effect in enhancing expression. Intron
sequences have been routinely incorporated into plant transformation
vectors, typically within the non-translated leader.

[0160] The present invention also relates to nucleic acid constructs
comprising one or more of the expression cassettes described above. The
construct can be a vector, such as a plant transformation vector. In one
embodiment, the vector is a plant transformation vector comprising a
polynucleotide comprising the sequence set forth in SEQ ID NO:34, 35, 36,
or 37.

Plants

[0161] As used herein, the term "plant part" or "plant tissue" includes
plant cells, plant protoplasts, plant cell tissue cultures from which
plants can be regenerated, plant calli, plant clumps, and plant cells
that are intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs,
husks, stalks, roots, root tips, anthers, and the like.

[0162] Plants useful in the present invention include plants that are
transgenic for at least a polynucleotide encoding a mutant HPPD
polypeptide or variant thereof that retains HPPD enzymatic activity,
alone or in combination with one or more additional nucleic acid
molecules encoding polypeptides that confer desirable traits. The type of
plant selected depends on a variety of factors, including for example,
the downstream use of the harvested plant material, amenability of the
plant species to transformation, and the conditions under which the
plants will be grown, harvested, and/or processed. One of skill will
further recognize that additional factors for selecting appropriate plant
varieties for use in the present invention include high yield potential,
good stalk strength, resistance to specific diseases, drought tolerance,
rapid dry down and grain quality sufficient to allow storage and shipment
to market with minimum loss.

[0163] Plants according to the present invention include any plant that is
cultivated for the purpose of producing plant material that is sought
after by man or animal for either oral consumption, or for utilization in
an industrial, pharmaceutical, or commercial process. The invention may
be applied to any of a variety of plants, including, but not limited to
maize, wheat, rice, barley, soybean, cotton, sorghum, beans in general,
rape/canola, alfalfa, flax, sunflower, safflower, millet, rye, sugarcane,
sugar beet, cocoa, tea, Brassica, cotton, coffee, sweet potato, flax,
peanut, clover; vegetables such as lettuce, tomato, cucurbits, cassaya,
potato, carrot, radish, pea, lentils, cabbage, cauliflower, broccoli,
Brussels sprouts, peppers, and pineapple; tree fruits such as citrus,
apples, pears, peaches, apricots, walnuts, avocado, banana, and coconut;
and flowers such as orchids, carnations and roses. Other plants useful in
the practice of the invention include perennial grasses, such as
switchgrass, prairie grasses, Indiangrass, Big bluestem grass and the
like. It is recognized that mixtures of plants may be used.

[0164] In addition, the term "crops" is to be understood as also including
crops that have been rendered tolerant to herbicides or classes of
herbicides (such as, for example, ALS inhibitors, for example
primisulfuron, prosulfuron and trifloxysulfuron, EPSPS
(5-enol-pyrovyl-shikimate-3-phosphate-synthase) inhibitors, GS (glutamine
synthetase) inhibitors) as a result of conventional methods of breeding
or genetic engineering. Examples of crops that have been rendered
tolerant to herbicides or classes of herbicides by genetic engineering
methods include glyphosate- and glufosinate-resistant crop varieties
commercially available under the trade names ROUNDUPREADY® and
LIBERTYLINK®. The method according to the present invention is
especially suitable for the protection of soybean crops which have also
been rendered tolerant to glyphosate and/or glufosinate and where HPPD
herbicides are used in a weed control programme along with other such
herbicides (glufosinate and/or glyphosate) for weed control.

[0165] It is further contemplated that the constructs of the invention may
be introduced into plant varieties having improved properties suitable or
optimal for a particular downstream use. For example, naturally-occurring
genetic variability results in plants with resistance or tolerance to
HPPD inhibitors or other herbicides, and such plants are also useful in
the methods of the invention. The method according to the present
invention can be further optimized by crossing the transgenes that
provide a level of tolerance, with soybean cultivars that exhibit an
enhanced level of tolerance to HPPD inhibitors that is found in a small
percentage of soybean lines.

Plant Transformation

[0166] Once an herbicide resistant or tolerant mutant HPPD polynucleotide,
alone or in combination with one or more additional nucleic acid
molecules encoding polypeptides that confer desirable traits, has been
cloned into an expression system, it is transformed into a plant cell.
The receptor and target expression cassettes of the present invention can
be introduced into the plant cell in a number of art-recognized ways. The
term "introducing" in the context of a polynucleotide, for example, a
nucleotide construct of interest, is intended to mean presenting to the
plant the polynucleotide in such a manner that the polynucleotide gains
access to the interior of a cell of the plant. Where more than one
polynucleotide is to be introduced, these polynucleotides can be
assembled as part of a single nucleotide construct, or as separate
nucleotide constructs, and can be located on the same or different
transformation vectors. Accordingly, these polynucleotides can be
introduced into the host cell of interest in a single transformation
event, in separate transformation events, or, for example, in plants, as
part of a breeding protocol. The methods of the invention do not depend
on a particular method for introducing one or more polynucleotides into a
plant, only that the polynucleotide(s) gains access to the interior of at
least one cell of the plant. Methods for introducing polynucleotides into
plants are known in the art including, but not limited to, transient
transformation methods, stable transformation methods, and virus-mediated
methods.

[0167] "Transient transformation" in the context of a polynucleotide is
intended to mean that a polynucleotide is introduced into the plant and
does not integrate into the genome of the plant.

[0168] By "stably introducing" or "stably introduced" in the context of a
polynucleotide introduced into a plant is intended the introduced
polynucleotide is stably incorporated into the plant genome, and thus the
plant is stably transformed with the polynucleotide.

[0169] "Stable transformation" or "stably transformed" is intended to mean
that a polynucleotide, for example, a nucleotide construct described
herein, introduced into a plant integrates into the genome of the plant
and is capable of being inherited by the progeny thereof, more
particularly, by the progeny of multiple successive generations.

[0170] Numerous transformation vectors available for plant transformation
are known to those of ordinary skill in the plant transformation arts,
and the genes pertinent to this invention can be used in conjunction with
any such vectors. The selection of vector will depend upon the preferred
transformation technique and the target species for transformation. For
certain target species, different antibiotic or herbicide selection
markers may be preferred. Selection markers used routinely in
transformation include the nptII gene, which confers resistance to
kanamycin and related antibiotics (Messing & Vierra Gene 19: 259-268
(1982); Bevan et al., Nature 304:184-187 (1983)), the pat and bar genes,
which confer resistance to the herbicide glufosinate (also called
phosphinothricin; see White et al., Nucl. Acids Res 18: 1062 (1990),
Spencer et al. Theor. Appl. Genet 79: 625-631 (1990) and U.S. Pat. Nos.
5,561,236 and 5,276,268), the hph gene, which confers resistance to the
antibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell. Biol. 4:
2929-2931), and the dhfr gene, which confers resistance to methatrexate
(Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), the EPSPS gene, which
confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and
5,188,642), the glyphosate N-acetyltransferase (GAT) gene, which also
confers resistance to glyphosate (Castle et al. (2004) Science,
304:1151-1154; U.S. Patent App. Pub. Nos. 20070004912, 20050246798, and
20050060767); and the mannose-6-phosphate isomerase gene, which provides
the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and
5,994,629). Alternatively, and in one preferred embodiment the HPPD gene
of the current invention is, in combination with the use of an HPPD
herbicide as selection agent, itself used as the selectable marker.

[0171] Methods for regeneration of plants are also well known in the art.
For example, Ti plasmid vectors have been utilized for the delivery of
foreign DNA, as well as direct DNA uptake, liposomes, electroporation,
microinjection, and microprojectiles. In addition, bacteria from the
genus Agrobacterium can be utilized to transform plant cells. Below are
descriptions of representative techniques for transforming both
dicotyledonous and monocotyledonous plants, as well as a representative
plastid transformation technique.

[0172] Many vectors are available for transformation using Agrobacterium
tumefaciens. These typically carry at least one T-DNA border sequence and
include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). For the
construction of vectors useful in Agrobacterium transformation, see, for
example, US Patent Application Publication No. 2006/0260011, herein
incorporated by reference. Transformation without the use of
Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences
in the chosen transformation vector and consequently vectors lacking
these sequences can be utilized in addition to vectors such as the ones
described above which contain T-DNA sequences. Transformation techniques
that do not rely on Agrobacterium include transformation via particle
bombardment, protoplast uptake (e.g. PEG and electroporation) and
microinjection. The choice of vector depends largely on the preferred
selection for the species being transformed. For the construction of such
vectors, see, for example, US Application No. 20060260011, herein
incorporated by reference.

[0173] For expression of a nucleotide sequence of the present invention in
plant plastids, plastid transformation vector pPH143 (WO 97/32011, See
Example 36) is used. The nucleotide sequence is inserted into pPH143
thereby replacing the PROTOX coding sequence. This vector is then used
for plastid transformation and selection of transformants for
spectinomycin resistance. Alternatively, the nucleotide sequence is
inserted in pPH143 so that it replaces the aadH gene. In this case,
transformants are selected for resistance to PROTOX inhibitors.

[0174] Transformation techniques for dicotyledons are well known in the
art and include Agrobacterium-based techniques and techniques that do not
require Agrobacterium. Non-Agrobacterium techniques involve the uptake of
exogenous genetic material directly by protoplasts or cells. This can be
accomplished by PEG or electroporation mediated uptake, particle
bombardment-mediated delivery, or microinjection. Examples of these
techniques are described by Paszkowski et al., EMBO J. 3: 2717-2722
(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et
al., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:
70-73 (1987). In each case the transformed cells are regenerated to whole
plants using standard techniques known in the art.

[0175] Agrobacterium-mediated transformation is a preferred technique for
transformation of dicotyledons because of its high efficiency of
transformation and its broad utility with many different species.
Agrobacterium transformation typically involves the transfer of the
binary vector carrying the foreign DNA of interest (e.g. pCIB200 or
pCIB2001) to an appropriate Agrobacterium strain which may depend of the
complement of vir genes carried by the host Agrobacterium strain either
on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for
pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). The
transfer of the recombinant binary vector to Agrobacterium is
accomplished by a triparental mating procedure using E. coli carrying the
recombinant binary vector, a helper E. coli strain which carries a
plasmid such as pRK2013 and which is able to mobilize the recombinant
binary vector to the target Agrobacterium strain. Alternatively, the
recombinant binary vector can be transferred to Agrobacterium by DNA
transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

[0176] Transformation of the target plant species by recombinant
Agrobacterium usually involves co-cultivation of the Agrobacterium with
explants from the plant and follows protocols well known in the art.
Transformed tissue is regenerated on selectable medium carrying the
antibiotic or herbicide resistance marker present between the binary
plasmid T-DNA borders.

[0177] Another approach to transforming plant cells with a gene involves
propelling inert or biologically active particles at plant tissues and
cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,
5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure
involves propelling inert or biologically active particles at the cells
under conditions effective to penetrate the outer surface of the cell and
afford incorporation within the interior thereof. When inert particles
are utilized, the vector can be introduced into the cell by coating the
particles with the vector containing the desired gene. Alternatively, the
target cell can be surrounded by the vector so that the vector is carried
into the cell by the wake of the particle. Biologically active particles
(e.g., dried yeast cells, dried bacterium or a bacteriophage, each
containing DNA sought to be introduced) can also be propelled into plant
cell tissue.

[0178] Transformation of most monocotyledon species has now also become
routine. Preferred techniques include direct gene transfer into
protoplasts using PEG or electroporation techniques, and particle
bombardment into callus tissue. Transformations can be undertaken with a
single DNA species or multiple DNA species (i.e. co-transformation) and
both of these techniques are suitable for use with this invention.
Co-transformation may have the advantage of avoiding complete vector
construction and of generating transgenic plants with unlinked loci for
the gene of interest and the selectable marker, enabling the removal of
the selectable marker in subsequent generations, should this be regarded
desirable. However, a disadvantage of the use of co-transformation is the
less than 100% frequency with which separate DNA species are integrated
into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

[0179] Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278
describe techniques for the preparation of callus and protoplasts from an
elite inbred line of maize, transformation of protoplasts using PEG or
electroporation, and the regeneration of maize plants from transformed
protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm
et al. (Biotechnology 8: 833-839 (1990)) have published techniques for
transformation of A188-derived maize line using particle bombardment.
Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200
(1993)) describe techniques for the transformation of elite inbred lines
of maize by particle bombardment. This technique utilizes immature maize
embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after
pollination and a PDS-1000He Biolistics device for bombardment.

[0181] Patent Application EP 0 332 581 describes techniques for the
generation, transformation and regeneration of Pooideae protoplasts.
These techniques allow the transformation of Dactylis and wheat.
Furthermore, wheat transformation has been described by Vasil et al.
(Biotechnology 10: 667-674 (1992)) using particle bombardment into cells
of type C long-term regenerable callus, and also by Vasil et al.
(Biotechnology 11:1553-1558 (1993)) and Weeks et al. (Plant Physiol.
102:1077-1084 (1993)) using particle bombardment of immature embryos and
immature embryo-derived callus. A preferred technique for wheat
transformation, however, involves the transformation of wheat by particle
bombardment of immature embryos and includes either a high sucrose or a
high maltose step prior to gene delivery. Prior to bombardment, any
number of embryos (0.75-1 mm in length) are plated onto MS medium with 3%
sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and
3 mg/l 2,4-D for induction of somatic embryos, which is allowed to
proceed in the dark. On the chosen day of bombardment, embryos are
removed from the induction medium and placed onto the osmoticum (i.e.
induction medium with sucrose or maltose added at the desired
concentration, typically 15%). The embryos are allowed to plasmolyze for
2-3 hours and are then bombarded. Twenty embryos per target plate is
typical, although not critical. An appropriate gene-carrying plasmid
(such as pCIB3064 or pSOG35) is precipitated onto micrometer size gold
particles using standard procedures. Each plate of embryos is shot with
the DuPont BIOLISTICS® helium device using a burst pressure of about
1000 psi using a standard 80 mesh screen. After bombardment, the embryos
are placed back into the dark to recover for about 24 hours (still on
osmoticum). After 24 hrs, the embryos are removed from the osmoticum and
placed back onto induction medium where they stay for about a month
before regeneration. Approximately one month later the embryo explants
with developing embryogenic callus are transferred to regeneration medium
(MS+1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate
selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l
methotrexate in the case of pSOG35). After approximately one month,
developed shoots are transferred to larger sterile containers known as
"GA7s" which contain half-strength MS, 2% sucrose, and the same
concentration of selection agent.

[0182] Transformation of monocotyledons using Agrobacterium has also been
described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of which
are incorporated herein by reference. See also, Negrotto et al., Plant
Cell Reports 19: 798-803 (2000), incorporated herein by reference.

[0183] For example, rice (Oryza sativa) can be used for generating
transgenic plants. Various rice cultivars can be used (Hiei et al., 1994,
Plant Journal 6:271-282; Dong et al., 1996, Molecular Breeding 2:267-276;
Hiei et al., 1997, Plant Molecular Biology, 35:205-218). Also, the
various media constituents described below may be either varied in
quantity or substituted. Embryogenic responses are initiated and/or
cultures are established from mature embryos by culturing on MS-CIM
medium (MS basal salts, 4.3 g/liter; B5 vitamins (200×), 5
ml/liter; Sucrose, 30 gaiter; proline, 500 mg/liter; glutamine, 500
mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml), 2 ml/liter;
adjust pH to 5.8 with 1 N KOH; Phytagel, 3 g/liter). Either mature
embryos at the initial stages of culture response or established culture
lines are inoculated and co-cultivated with the Agrobacterium tumefaciens
strain LBA4404 (Agrobacterium) containing the desired vector
construction. Agrobacterium is cultured from glycerol stocks on solid YPC
medium (100 mg/L spectinomycin and any other appropriate antibiotic) for
about 2 days at 28° C. Agrobacterium is re-suspended in liquid
MS-CIM medium. The Agrobacterium culture is diluted to an OD600 of
0.2-0.3 and acetosyringone is added to a final concentration of 200 uM.
Acetosyringone is added before mixing the solution with the rice cultures
to induce Agrobacterium for DNA transfer to the plant cells. For
inoculation, the plant cultures are immersed in the bacterial suspension.
The liquid bacterial suspension is removed and the inoculated cultures
are placed on co-cultivation medium and incubated at 22° C. for
two days. The cultures are then transferred to MS-CIM medium with
Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium. For
constructs utilizing the PMI selectable marker gene (Reed et al., In
Vitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred to
selection medium containing Mannose as a carbohydrate source (MS with 2%
Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4
weeks in the dark. Resistant colonies are then transferred to
regeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1
mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) and
grown in the dark for 14 days. Proliferating colonies are then
transferred to another round of regeneration induction media and moved to
the light growth room. Regenerated shoots are transferred to GA7
containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2
weeks and then moved to the greenhouse when they are large enough and
have adequate roots. Plants are transplanted to soil in the greenhouse
(To generation) grown to maturity, and the T1 seed is harvested.

[0184] The plants obtained via transformation with a nucleic acid sequence
of interest in the present invention can be any of a wide variety of
plant species, including those of monocots and dicots; however, the
plants used in the method of the invention are preferably selected from
the list of agronomically important target crops set forth elsewhere
herein. The expression of a gene of the present invention in combination
with other characteristics important for production and quality can be
incorporated into plant lines through breeding. Breeding approaches and
techniques are known in the art. See, for example, Welsh J. R.,
Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY
(1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy
Madison, Wis. (1983); Mayo O., The Theory of Plant Breeding, Second
Edition, Clarendon Press, Oxford (1987); Singh, D. P., Breeding for
Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and
Wricke and Weber, Quantitative Genetics and Selection Plant Breeding,
Walter de Gruyter and Co., Berlin (1986).

[0186] The genetic properties engineered into the transgenic seeds and
plants described above are passed on by sexual reproduction or vegetative
growth and can thus be maintained and propagated in progeny plants.
Generally, maintenance and propagation make use of known agricultural
methods developed to fit specific purposes such as tilling, sowing or
harvesting.

[0187] Use of the advantageous genetic properties of the transgenic plants
and seeds according to the invention can further be made in plant
breeding. Depending on the desired properties, different breeding
measures are taken. The relevant techniques are well known in the art and
include but are not limited to hybridization, inbreeding, backcross
breeding, multi-line breeding, variety blend, interspecific
hybridization, aneuploid techniques, etc. Thus, the transgenic seeds and
plants according to the invention can be used for the breeding of
improved plant lines that, for example, increase the effectiveness of
conventional methods such as herbicide or pesticide treatment or allow
one to dispense with said methods due to their modified genetic
properties.

[0188] Many suitable methods for transformation using suitable selection
markers such as kanamycin, binary vectors such as from Agrobacterium and
plant regeneration as, for example, from tobacco leaf discs are well
known in the art. Optionally, a control population of plants are likewise
transformed with a polynucleotide expressing the control HPPD.
Alternatively, an untransformed dicot plant such as Arabidopsis or
Tobacco can be used as a control since this, in any case, expresses its
own endogenous HPPD.

Herbicide Resistance

[0189] The present invention provides transgenic plants, plant cells,
tissues, and seeds that have been transformed with a nucleic acid
molecule encoding a mutant HPPD or variant thereof that confers
resistance or tolerance to herbicides, alone or in combination with one
or more additional nucleic acid molecules encoding polypeptides that
confer desirable traits.

[0190] In one embodiment, the transgenic plants of the invention exhibit
resistance or tolerance to application of herbicide in an amount of from
about 5 to about 2,000 grams per hectare (g/ha), including, for example,
about 5 g/ha, about 10 g/ha, about 15 g/ha, about 20 g/ha, about 25 g/ha,
about 30 g/ha, about 35 g/ha, about 40 g/ha, about 45 g/ha, about 50
g/ha, about 55 g/ha, about 60 g/ha, about 65 g/ha, about 70 g/ha, about
75 g/ha, about 80 g/ha, about 85 g/ha, about 90 g/ha, about 95 g/ha,
about 100 g/ha, about 110 g/ha, about 120 g/ha, about 130 g/ha, about 140
g/ha, about 150 g/ha, about 160 g/ha, about 170 g/ha, about 180 g/ha,
about 190 g/ha, about 200 g/ha, about 210 g/ha, about 220 g/ha, about 230
g/ha, about 240 g/ha, about 250 g/ha, about 260 g/ha, about 270 g/ha,
about 280 g/ha, about 290 g/ha, about 300 g/ha, about 310 g/ha, about 320
g/ha, about 330 g/ha, about 340 g/ha, about 350 g/ha, about 360 g/ha,
about 370 g/ha, about 380 g/ha, about 390 g/ha, about 400 g/ha, about 410
g/ha, about 420 g/ha, about 430 g/ha, about 440 g/ha, about 450 g/ha,
about 460 g/ha, about 470 g/ha, about 480 g/ha, about 490 g/ha, about 500
g/ha, about 510 g/ha, about 520 g/ha, about 530 g/ha, about 540 g/ha,
about 550 g/ha, about 560 g/ha, about 570 g/ha, about 580 g/ha, about 590
g/ha, about 600 g/ha, about 610 g/ha, about 620 g/ha, about 630 g/ha,
about 640 g/ha, about 650 g/ha, about 660 g/ha, about 670 g/ha, about 680
g/ha, about 690 g/ha, about 700 g/ha, about 710 g/ha, about 720 g/ha,
about 730 g/ha, about 740 g/ha, about 750 g/ha, about 760 g/ha, about 770
g/ha, about 780 g/ha, about 790 g/ha, about 800 g/ha, about 810 g/ha,
about 820 g/ha, about 830 g/ha, about 840 g/ha, about 850 g/ha, about 860
g/ha, about 870 g/ha, about 880 g/ha, about 890 g/ha, about 900 g/ha,
about 910 g/ha, about 920 g/ha, about 930 g/ha, about 940 g/ha, about 950
g/ha, about 960 g/ha, about 970 g/ha, about 980 g/ha, about 990 g/ha,
about 1,000, g/ha, about 1,010 g/ha, about 1,020 g/ha, about 1,030 g/ha,
about 1,040 g/ha, about 1,050 g/ha, about 1,060 g/ha, about 1,070 g/ha,
about 1,080 g/ha, about 1,090 g/ha, about 1,100 g/ha, about 1,110 g/ha,
about 1,120 g/ha, about 1,130 g/ha, about 1,140 g/ha, about 1,150 g/ha,
about 1,160 g/ha, about 1,170 g/ha, about 1,180 g/ha, about 1,190 g/ha,
about 1,200 g/ha, about 1,210 g/ha, about 1,220 g/ha, about 1,230 g/ha,
about 1,240 g/ha, about 1,250 g/ha, about 1,260 g/ha, about 1,270 g/ha,
about 1,280 g/ha, about 1,290 g/ha, about 1,300 g/ha, about 1,310 g/ha,
about 1,320 g/ha, about 1,330 g/ha, about 1,340 g/ha, about 1,350 g/ha,
about 360 g/ha, about 1,370 g/ha, about 1,380 g/ha, about 1,390 g/ha,
about 1,400 g/ha, about 1,410 g/ha, about 1,420 g/ha, about 1,430 g/ha,
about 1,440 g/ha, about 1,450 g/ha, about 1,460 g/ha, about 1,470 g/ha,
about 1,480 g/ha, about 1,490 g/ha, about 1,500 g/ha, about 1,510 g/ha,
about 1,520 g/ha, about 1,530 g/ha, about 1,540 g/ha, about 1,550 g/ha,
about 1,560 g/ha, about 1,570 g/ha, about 1,580 g/ha, about 1,590 g/ha,
about 1,600 g/ha, about 1,610 g/ha, about 1,620 g/ha, about 1,630 g/ha,
about 1,640 g/ha, about 1,650 g/ha, about 1,660 g/ha, about 1,670 g/ha,
about 1,680 g/ha, about 1,690 g/ha, about 1,700 g/ha, about 1,710 g/ha,
about 1,720 g/ha, about 1,730 g/ha, about 1,740 g/ha, about 1,750 g/ha,
about 1,760 g/ha, about 1,770 g/ha, about 1,780 g/ha, about 1,790 g/ha,
about 1,800 g/ha, about 1,810 g/ha, about 1,820 g/ha, about 1,830 g/ha,
about 1,840 g/ha, about 1,850 g/ha, about 1,860 g/ha, about 1,870 g/ha,
about 1,880 g/ha, about 1,890 g/ha, about 1,900 g/ha, about 1,910 g/ha,
about 1,920 g/ha, about 1,930 g/ha, about 1,940 g/ha, about 1,950 g/ha,
about 1,960 g/ha, about 1,970 g/ha, about 1,980 g/ha, about 1,990 g/ha,
or about 2,000.

[0191] The average and distribution of herbicide tolerance or resistance
levels of a range of primary plant transformation events are evaluated in
the normal manner based upon plant damage, meristematic bleaching
symptoms etc. at a range of different concentrations of herbicides. These
data can be expressed in terms of, for example, GR50 values derived from
dose/response curves having "dose" plotted on the x-axis and "percentage
kill", "herbicidal effect", "numbers of emerging green plants" etc.
plotted on the y-axis where increased GR50 values correspond to increased
levels of inherent inhibitor-tolerance (e.g. increased Ki/KmHPP
value) and/or level of expression of the expressed HPPD polypeptide.

[0192] The methods of the present invention are especially useful to
protect crops from the herbicidal injury of HPPD inhibitor herbicides of
the classes of HPPD chemistry described below. In one embodiment, the
selected from the group consisting of:

[0193] a) a compound of formula (Ia)

##STR00001##

wherein R1 and R2 are hydrogen or together form an ethylene
bridge; R3 is hydroxy or phenylthio-; R4 is halogen, nitro,
C1-C4alkyl, C1-C4alkoxy-C1-C4alkyl-,
C1-C4alkoxy-C1-C4alkoxy-C1-C4alkyl-; X is
methine, nitrogen, or C--R5 wherein R5 is hydrogen,
C1-C4alkoxy, C1-C4haloalkoxy-C1-C4alkyl-,
or a group

##STR00002##

and R6 is C1-C4alkylsulfonyl- or C1-C4haloalkyl;

[0194] b) a compound of formula (Ib)

##STR00003##

R1 and R2 are independently C1-C4alkyl; and the free
acids thereof;

[0195] c) a compound of formula (Ic)

##STR00004##

wherein R1 is hydroxy, phenylcarbonyl-C1-C4alkoxy- or
phenylcarbonyl-C1-C4alkoxy- wherein the phenyl moiety is
substituted in para-position by halogen or C1-C4alkyl, or
phenylsulfonyloxy- or phenylsulfonyloxy- wherein the phenyl moiety is
substituted in para-position by halogen or C1-C4alkyl; R2
is C1-C4alkyl; R3 is hydrogen or C1-C4alkyl;
R4 and R6 are independently halogen, C1-C4alkyl,
C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and R5
is hydrogen, C1-C4alkyl,
C1-C4alkoxy-C1-C4alkoxy-, or a group

##STR00005##

[0196] d) a compound of formula (Id)

##STR00006##

wherein R1 is hydroxy; R2 is C1-C4alkyl; R3 is
hydrogen; and R4, R5 and R6 are independently
C1-C4alkyl;

[0197] e) a compound of formula (Ie)

##STR00007##

wherein R1 is cyclopropyl; R2 and R4 are independently
halogen, C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and
R3 is hydrogen;

[0198] f) a compound of formula (If)

##STR00008##

wherein R1 is cyclopropyl; R2 and R4 are independently
halogen, C1-C4haloalkyl, or C1-C4alkylsulfonyl-; and
R3 is hydrogen;

[0199] g) a compound of formula (Ig) or Formula (Ih)

##STR00009##

wherein: R2 is selected from the group consisting of
C1-C3alkyl, C1-C3haloalkyl,
C1-C3alkoxy-C1-C3 alkyl and C1-C3
alkoxy-C2-C3alkoxy-C1-C3-alkyl; R5 is hydrogen
or methyl; R6 is selected from the group consisting of hydrogen,
fluorine, chlorine, hydroxyl and methyl; R7 is selected from the
group consisting of hydrogen, halogen, hydroxyl, sulfhydryl,
C1-C6alkyl, C3-C6cycloalkyl,
C1-C6haloalkyl, C2-C6haloalkenyl,
C2-C6alkenyl, C3-C6alkynyl, C1-C6alkoxy,
C4-C7cycloalkoxy, C1-C6haloalkoxy,
C1-C6alkylthio, C1-C6alkylsulfinyl,
C1-C6alkylsulfonyl, C1-C6haloalkylthio, amino,
C1-C6alkylamino, C2-C6dialkylamino,
C2-C6dialkylaminosulfonyl, C1-C6alkylaminosulfonyl,
C1-C6alkoxy-C1-C6alkyl,
C1-C6alkoxy-C2-C6alkoxy,
C1-C6alkoxy-C2-C6 alkoxy-C1-C6-alkyl,
C3-C6alkenyl-C2-C6alkoxy,
C3-C6alkynyl-C1-C6alkoxy,
C1-C6alkoxycarbonyl, C1-C6alkylcarbonyl,
C1-C4alkylenyl-S(O)p-R', C1-C4alkylenyl-CO2--R',
C1-C4alkylenyl-(CO)N--R'R', phenyl, phenylthio, phenylsulfinyl,
phenylsulfonyl, phenoxy, pyrrolidinyl, piperidinyl, morpholinyl and 5 or
6-membered heteroaryl or heteroaryloxy, the heteroaryl containing one to
three heteroatoms, each independently selected from the group consisting
of oxygen, nitrogen and sulphur, wherein the phenyl or heteroaryl
component may be optionally substituted by a substituent selected from
the group consisting of C1-C3alkyl, C1-C3haloalkyl,
C1-C3 alkoxy, C1-C3haloalkoxy, halo, cyano, and
nitro;

X=O or S;

[0200] n=0 or 1; m=0 or 1 with the proviso that if m=1 then n=0 and if n=1
then m=0; p=0, 1, or 2; R' is independently selected from the group
consisting of hydrogen and C1-C6alkyl; R8 is selected from
the group consisting of hydrogen, C1-C6alkyl,
C1-C6haloalkyl,
C1-C6alkylcarbonyl-C1-C3alkyl,
C3-C6cycloalkylalkeneyl for example cyclohexylmethylenyl,
C3-C6alkynylalkyleneyl for example propargyl,
C2-C6-alkenylalkylenyl for example allyl, C1-C6alkoxy
C1-C6alkyl, cyano-C1-C6-alkyl,
arylcarbonyl-C1-C3-alkyl (wherein the aryl may be optionally
substituted with a substituent selected from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), aryl-C1-C6alkyl (wherein the aryl may be optionally
substituted with a substituent selected from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), C1-C6alkoxy C1-C6alkoxy
C1-C6alkyl and a 5 or 6-membered
heteroaryl-C1-C3-alkyl or heterocyclyl-C1-C3-alkyl,
the heteroaryl or heterocyclyl containing one to three heteroatoms, each
independently selected from the group consisting of oxygen, nitrogen and
sulphur, wherein the heterocyclyl or heteroaryl component may be
optionally substituted by a substituent selected from the group
consisting of halo, C1-C3alkyl, C1-C3haloalkyl, and
C1-C3 alkoxy; Q is selected from the group consisting of:

##STR00010##

wherein A1 is selected from the group consisting of O, C(O), S, SO,
SO2 and (CReRf)q; q=0, 1 or 2; Ra, Rb,
Rc, Rd, Re and Rf are each independently selected
from the group consisting of C1-C4alkyl which may be mono-, di-
or tri-substituted by substituents selected from the group consisting of
C1-C4alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylthio,
C1-C4alkylsulfinyl, C1-C4alkylsulfonyl,
C1-C4alkylcarbonyl, phenyl and heteroaryl, it being possible
for the phenyl and heteroaryl groups in turn to be mono-, di- or
tri-substituted by substituents selected from the group consisting of
C1-C4alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylsulfonyl and
C1-C4haloalkyl, the substituents on the nitrogen in the
heterocyclic ring being other than halogen; or Ra, Rb, Rc,
Rd, Re and Rf are each independently selected from the
group consisting of hydrogen, C1-C4alkoxy, halogen, hydroxy,
cyano, hydroxycarbonyl, C1-C4alkoxycarbonyl,
C1-C4alkylthio, C1-C4alkylsulfinyl,
C1-C4alkylsulfonyl, C1-C4alkylcarbonyl, phenyl or
heteroaryl, it being possible for the phenyl and heteroaryl groups in
turn to be mono-, di- or tri-substituted by substituents selected from
the group consisting of C1-C4alkoxy, halogen, hydroxy, cyano,
hydroxycarbonyl, C1-C4alkoxycarbonyl,
C1-C4alkylsulfonyl and C1-C4haloalkyl, the
substituents on the nitrogen in the heterocyclic ring being other than
halogen; or Ra and Rb together form a 3- to 5-membered
carbocyclic ring which may be substituted by C1-C4alkyl and may
be interrupted by oxygen, sulfur, S(O), SO2, OC(O), NRg or by
C(O); or Ra and Rc together form a C1-C3alkylene
chain which may be interrupted by oxygen, sulfur, SO, SO2, OC(O),
NRh or by C(O); it being possible for that C1-C3alkylene
chain in turn to be substituted by C1-C4alkyl; Rg and
Rh are each independently of the other C1-C4alkyl,
C1-C4haloalkyl, C1-C4alkylsulfonyl,
C1-C4alkylcarbonyl or C1-C4alkoxycarbonyl; Ri is
C1-C4alkyl; R3 is selected from the group consisting of
C1-C6alkyl, optionally substituted with halogen and/or
C1-C3alkoxy; and C3-C6 cycloalkyl optionally
substituted with halogen and/or C1-C3alkoxy; R9 is
selected from the group consisting of cyclopropyl, CF3 and i.-Pr;
R10 is selected from the group consisting of hydrogen, I, Br,
SR11, S(O)R11, S(O)2R11 and CO2R11; and
R11 is C1-4 alkyl; h) a compound of formula (Ij), (Ik), or (Im)

##STR00011##

or an agronomically acceptable salt of said compound, wherein: R1 is
selected from the group consisting of hydrogen, C1-C6alkyl,
C1-C6haloalkyl, C1-C3alkoxy-C1-C3 alkyl,
C1-C3 alkoxy-C1-C3alkoxy-C1-C3-alkyl,
C1-C3alkoxy-C1-C3-haloalkyl,
C1-C3-alkoxy-C1-C3-alkoxy-C1-C3-haloalkyl,
C4-C6-oxasubstituted cycloalkoxy-C1-C3-alkyl,
C4-C6-oxasubstituted
cycloalkyl-C1-C3-alkoxy-C1-C3-alkyl,
C4-C6-oxasubstituted cycloalkoxy-C1-C3-haloalkyl,
C4-C6-oxasubstituted
cycloalkyl-C1-C3-alkoxy-C1-C3-halo alkyl,
(C1-C3 alkanesulfonyl-C1-C3
alkylamino)-C1-C3 alkyl, (C1-C3
alkanesulfonyl-C3-C4 cycloalkylamino)-C1-C3alkyl,
C1-C6alkylcarbonyl-C1-C3alkyl,
C3-C6cycloalkyl-C2-C6alkenyl, C3-C6alkynyl,
C2-C6-alkenyl, cyano-C1-C6-alkyl,
arylcarbonyl-C1-C3-alkyl (wherein the aryl may be optionally
substituted with one or more substituents from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), aryl-C1-C6alkyl (wherein the aryl may be optionally
substituted with one or more substituents from the group consisting of
halo, C1-C3-alkoxy, C1-C3-alkyl, C1-C3
haloalkyl), aryl, 5 or 6-membered heteroaryl, 5 or 6-membered
heteroaryl-C1-C3alkyl and heterocyclyl-C1-C3alkyl,
the heteroaryl or heterocyclyl containing one to three heteroatoms each
independently selected from the group consisting of oxygen, nitrogen and
sulphur, and wherein the aryl, heterocyclyl or heteroaryl component may
be optionally substituted by one or more substituents selected from the
group consisting of halo, C1-C3alkyl, C1-C3haloalkyl,
C1-C3 alkoxy, C1-C3 haloalkoxy,
C1-C6alkyl-S(O)p-, C1-C6haloalkyl-S(O)p-, cyano and
nitro; R5 is selected from the group consisting of hydrogen, chloro,
fluoro and methyl; R6 is selected from the group consisting of
hydrogen, fluorine, chlorine, hydroxyl and methyl; R7 is selected
from the group consisting of hydrogen, cyano, nitro, halogen, hydroxyl,
sulfhydryl, C1-C6alkyl, C3-C6cycloalkyl,
C1-C6haloalkyl, C2-C6haloalkenyl,
C2-C6alkenyl, aryl-C2-C6alkenyl,
C3-C6alkynyl, C1-C6alkoxy, C4-C7
cycloalkoxy, C1-C6haloalkoxy, C1-C6alkyl-S(O)p,
C3-C6cycloalkyl-S(O)pC1-C6haloalkyl-S(O)p,
C3-C6 halocycloalkyl-S(O)p, C1-C6alkylcarbonylamino,
(C1-C6alkylcarbonyl)C1-C3alkylamino,
(C3-C6cycloalkylcarbonyl)amino,
(C3-C6cycloalkylcarbonyl)C1-C3alkylamino,
arylcarbonylamino, (arylcarbonyl)-C1-3alkylamino,
(heteroarylcarbonyl)amino, (heteroarylcarbonyl)C1-C3alkylamino,
amino, C1-C6alkylamino, C2-C6dialkylamino,
C2-C6alkenylamino,
C1-C6alkoxy-C2-C6-alkylamino,
(C1-C6alkoxy-C2-C4-alkyl)-C1-C6-alkylamino,
C3-C6 cycloalkylamino, C3-C6 cyclohaloalkylamino,
C1-C3alkoxy-C3-C6 cycloalkylamino, C3-C6
alkynylamino, dialkylamino in which the substituents join to form a 4-6
membered ring (e.g. pyrrolidinyl, piperidinyl) optionally containing
oxygen (e.g. morpholinyl) and/or optionally substituted by
C1-C3-alkoxy and/or halogen (especially fluorine),
C2-C6dialkylaminosulfonyl, C1-C6alkylaminosulfonyl,
C1-C6alkoxy-C1-C6alkyl,
C1-C6alkoxy-C2-C6alkoxy,
C1-C6alkoxy-C2-C6 alkoxy-C1-C6-alkyl,
C3-C6alkenyl-C2-C6alkoxy,
C3-C6alkynyl-C1-C6alkoxy,
C1-C6alkoxycarbonyl, C1-C6alkylcarbonyl,
C1-C4alkylenyl-S(O)p--R',
C1-C4alkylenyl-CO2--R',
C1-C4alkylenyl-(CO)N--R'R', aryl (e.g. phenyl), aryl
C1-C3alkyl, aryl-S(O)p, heteroaryl-S(O)p, aryloxy (e.g.
phenoxy), a 5 or 6-membered heteroaryl, heteroaryl C1-C3 alkyl
and heteroaryloxy, the heteroaryl containing one to three heteroatoms,
each independently selected from the group consisting of oxygen, nitrogen
and sulphur, wherein the aryl or heteroaryl component may be optionally
substituted by one or more substituents selected from the group
consisting of C1-C3alkyl, C1-C3haloalkyl,
C1-C3 alkoxy, C1-C3haloalkoxy, halo, cyano and nitro;

X1=N--(O)n or C--R8;

X2=O or S;

[0201] n=0 or 1; p=0, 1 or 2; R' is independently selected from the group
consisting of hydrogen and C1-C6alkyl; R8 is selected from
the group consisting of hydrogen, halogen, C1-C6alkyl,
C1-C6haloalkyl,
C1-C6alkylcarbonyl-C1-C3alkyl,
C3-C6cycloalkyl-C2-C6alkenyl for example
cyclohexylmethylenyl, C3-C6alkynyl (for example propargyl),
C2-C6-alkenyl (for example allyl), C1-C6alkoxy
C1-C6alkyl, cyano-C1-C6-alkyl,
arylcarbonyl-C1-C3-alkyl (wherein the aryl may be optionally
substituted with one or more substituents selected from the group
consisting of halo, C1-C3-alkoxy, C1-C3-alkyl,
C1-C3 haloalkyl), aryl-C1-C6alkyl (wherein the aryl
may be optionally substituted with one or more substituents from the
group consisting of halo, C1-C3-alkoxy, C1-C3-alkyl,
C1-C3 haloalkyl), C1-C6alkoxyC1-C6alkoxy
C1-C6alkyl, aryl, a 5 or 6-membered heteroaryl, a 5 or
6-membered heteroaryl-C1-C3-alkyl and
heterocyclyl-C1-C3-alkyl, the heteroaryl or heterocyclyl
containing one to three heteroatoms each independently selected from the
group consisting of oxygen, nitrogen and sulphur, and wherein the aryl,
heterocyclyl or heteroaryl component may be optionally substituted by one
or more substituents from the group consisting of halogen,
C1-C3alkyl, C1-C3haloalkyl and C1-C3
alkoxy, cyano and nitro; Q is selected from the group consisting of: --

##STR00012##

wherein A1 is selected from the group consisting of O, C(O), S, SO,
SO2 and (CReRf)q; q=0, 1 or 2; Ra, Rb,
Rc, Rd, Re and Rf are each independently selected
from the group consisting of C1-C4alkyl which may be mono-, di-
or tri-substituted by substituents selected from the group consisting of
C1-C4alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylthio,
C1-C4alkylsulfinyl, C1-C4alkylsulfonyl,
C1-C4alkylcarbonyl, phenyl and heteroaryl, it being possible
for the phenyl and heteroaryl groups in turn to be mono-, di- or
tri-substituted by substituents selected from the group consisting of
C1-C4alkoxy, halogen, hydroxy, cyano, hydroxycarbonyl,
C1-C4alkoxycarbonyl, C1-C4alkylsulfonyl and
C1-C4haloalkyl, the substituents on the nitrogen in the
heterocyclic ring being other than halogen; or Ra, Rb, Rc,
Rd, Re and Rf are each independently selected from the
group consisting of hydrogen, C1-C4alkoxy, halogen, hydroxy,
cyano, hydroxycarbonyl, C1-C4alkoxycarbonyl,
C1-C4alkylthio, C1-C4alkylsulfinyl,
C1-C4alkylsulfonyl, C1-C4alkylcarbonyl, phenyl or
heteroaryl, it being possible for the phenyl and heteroaryl groups in
turn to be mono-, di- or tri-substituted by substituents selected from
the group consisting of C1-C4alkoxy, halogen, hydroxy, cyano,
hydroxycarbonyl, C1-C4alkoxycarbonyl,
C1-C4alkylsulfonyl and C1-C4haloalkyl, the
substituents on the nitrogen in the heterocyclic ring being other than
halogen; or Ra and Rb together form a 3- to 5-membered
carbocyclic ring which may be substituted by C1-C4alkyl and may
be interrupted by oxygen, sulfur, S(O), SO2, OC(O), NR9 or by
C(O); or Ra and Re together form a C1-C3alkylene
chain which may be interrupted by oxygen, sulfur, SO, SO2, OC(O),
NRh or by C(O); it being possible for that C1-C3alkylene
chain in turn to be substituted by C1-C4alkyl; Rg and
Rh are each independently of the other C1-C4alkyl,
C1-C4haloalkyl, C1-C4alkylsulfonyl,
C1-C4alkylcarbonyl or C1-C4alkoxycarbonyl; Ri is
C1-C4alkyl; Rj is selected from the group consisting of
hydrogen, C1-C4 alkyl and C3-C6 cycloalkyl; R3
is selected from the group consisting of C1-C6alkyl, optionally
substituted with halogen and/or C1-C3alkoxy, and
C3-C6 cycloalkyl optionally substituted with halogen and/or
C1-C3alkoxy; R9 is selected from the group consisting of
cyclopropyl, CF3 and i.-Pr; R10 is selected from the group
consisting of hydrogen, I, Br, SR11, S(O)R11,
S(O)2R11 and CO2R11; and R11 is C1-4 alkyl.

[0202] With respect to the structures (Ia)-(Im) described herein:

[0203] Halogen encompasses fluorine, chlorine, bromine or iodine. The same
correspondingly applies to halogen in the context of other definitions,
such as haloalkyl or halophenyl.

[0206] Suitable haloalkenyl radicals include alkenyl groups substituted
one or more times by halogen, halogen being fluorine, chlorine, bromine
or iodine and especially fluorine or chlorine, for example
2,2-difluoro-1-methylvinyl, 3-fluoropropenyl, 3-chloropropenyl,
3-bromopropenyl, 2,3,3-trifluoropropenyl, 2,3,3-trichloropropenyl and
4,4,4-trifluorobut-2-en-1-yl. Preferred C2-C6alkenyl radicals
substituted once, twice or three times by halogen are those having a
chain length of from 2 to 5 carbon atoms. Suitable haloalkylalkynyl
radicals include, for example, alkylalkynyl groups substituted one or
more times by halogen, halogen being bromine or iodine and, especially,
fluorine or chlorine, for example 3-fluoropropynyl,
5-chloropent-2-yn-1-yl, 5-bromopent-2-yn-1-yl, 3,3,3-trifluoropropynyl
and 4,4,4-trifluoro-but-2-yn-1-yl. Preferred alkylalkynyl groups
substituted one or more times by halogen are those having a chain length
of from 3 to 5 carbon atoms.

[0215] Cycloalkyl groups preferably have from 3 to 6 ring carbon atoms and
may be substituted by one or more methyl groups; they are preferably
unsubstituted, for example cyclopropyl, cyclobutyl, cyclopentyl or
cyclohexyl.

[0216] Phenyl, including phenyl as part of a substituent such as phenoxy,
benzyl, benzyloxy, benzoyl, phenylthio, phenylalkyl, phenoxyalkyl or
tosyl, may be in mono- or poly-substituted form, in which case the
substituents may, as desired, be in the ortho-, meta- and/or
para-position(s).

[0218] Heteroaryl, including heteroaryl as part of a substituent such as
heteroaryloxy, means a five or six member heteroaryl containing one to
three heteroatoms, each independently selected from the group consisting
of oxygen, nitrogen and sulphur. It should be understood that the
heteroaryl component may be optionally mono or poly substituted. The term
heteroaryl thus includes, for example, furanyl, thiopheneyl, thiazolyl,
oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, isothiazolyl, pyridyl,
pyridazinyl, pyrazinyl, pyrimidinyl, triazolyl.

[0219] Compounds of Formula Ij may contain asymmetric centres and may be
present as a single enantiomer, pairs of enantiomers in any proportion
or, where more than one asymmetric centre are present, contain
diastereoisomers in all possible ratios. Typically one of the enantiomers
has enhanced biological activity compared to the other possibilities.

[0220] Similarly, where there are disubstituted alkenes, these may be
present in E or Z form or as mixtures of both in any proportion.

[0221] Furthermore, compounds of Formula Ij comprising Q1, Q5, Q6 or Q7 or
when R1 is hydrogen may be in equilibrium with alternative hydroxyl
tautomeric forms. It should be appreciated that all tautomeric forms
(single tautomer or mixtures thereof), racemic mixtures and single
isomers are included within the scope of the present invention.

[0222] The skilled person will also appreciate that if n is 1 with regard
to Formula Ij to form the N-oxide then the nitrogen and oxygen will be
charged accordingly (N+O.sup.-).

[0223] In a preferred embodiment of the present invention X2 is
oxygen.

[0224] In another preferred embodiment R1 is selected from the group
consisting of hydrogen, C1-C6alkyl,
C1-C3alkoxyC1-C3alkyl, C1-C3alkoxy
C2-C3alkoxyC1-C3alkyl, C1-C6haloalkyl,
C1-C3alkoxy-C1-C3haloalkyl and phenyl.

[0225] In another preferred embodiment R1 is aryl, preferably phenyl,
or a 5 or 6-membered heteroaryl containing one to three heteroatoms each
independently selected from the group consisting of oxygen, nitrogen and
sulphur, and wherein the aryl or heteroaryl may be optionally substituted
by one or more substituents selected from the group consisting of halo,
C1-C3alkyl, C1-C3haloalkyl, C1-C3 alkoxy,
C1-C3 haloalkoxy, C1-C6alkyl-S(O)p-,
C1-C6haloalkyl-S(O)p-, cyano and nitro.

[0226] In another preferred embodiment R5 is hydrogen.

[0227] In another preferred embodiment R6 is hydrogen or fluorine.

[0228] In another preferred embodiment Rj is selected from the group
consisting of hydrogen, methyl and cyclopropyl.

[0229] In another preferred embodiment the herbicidal compound is of
Formula (Ik):

##STR00013##

[0230] In a more preferred embodiment of the present invention the
herbicidal compound is of Formula (Ik) wherein Q is Q1, in particular
wherein A1 is CReRf and wherein Ra, Rb, Rc,
Rd, Re and Rf are hydrogen, and wherein q=1. In another
preferred embodiment of the present invention Q is Q1, wherein A1 is
CReRf and wherein, Rb, Rd, Re and Rf are
hydrogen, Ra and Rc together form an ethylene chain and wherein
q=1

[0232] In another preferred embodiment the herbicidal compound is of
Formula (Im):

##STR00014##

[0233] In another preferred embodiment of the present invention the
herbicidal compound is of Formula (Im), wherein Q is Q1, in particular
wherein A1 is CReRf and wherein Ra, Rb, Rc,
Rd, Re and Rf are hydrogen, and wherein q=1. In another
preferred embodiment of the present invention Q is Q1, wherein A1 is
CReRf and wherein, Rb, Rd, Re and Rf are
hydrogen, Ra and Re together form an ethylene chain and wherein
q=1.

[0238] The level of expression of the mutant HPPD should be sufficient to
reduce substantially (relative to likewise treated plants but lacking the
mutant HPPD transgenes) the residue level of parent herbicide throughout
the plant tissue. One of ordinary skill in the art will of course
understand that certain mutant HPPD enzymes may confer resistance to
certain subgroups of HPPD chemistry, and one enzyme may not provide
resistance to all HPPDs.

Methods of Use

[0239] The present invention further provides a method of selectively
controlling weeds at a locus comprising crop plants and weeds, wherein
the plants are obtained by any of the methods of the current invention
described above, wherein the method comprises application to the locus of
a weed controlling amount of one or more herbicides. Any of the
transgenic plants described herein may be used within these methods of
the invention. The term "locus" may include soil, seeds, and seedlings,
as well as established vegetation. Herbicides can suitably be applied
pre-emergence or post-emergence of the crop or weeds.

[0240] The term "weed controlling amount" is meant to include
functionally, an amount of herbicide which is capable of affecting the
growth or development of a given weed. Thus, the amount may be small
enough to simply retard or suppress the growth or development of a given
weed, or the amount may be large enough to irreversibly destroy a given
weed.

[0241] Thus, the present invention provides a method of controlling weeds
at a locus comprising applying to the locus a weed-controlling amount of
one or more herbicides, where the locus comprises a transgenic plant that
has been transformed with a nucleic acid molecule encoding a mutant HPPD
polypeptide or variant thereof that confers resistance or tolerance to
HPPD herbicides, alone or in combination with one or more additional
nucleic acid molecules encoding polypeptides that confer desirable
traits. In one embodiment, the desirable trait is resistance or tolerance
to an herbicide, including, for example, herbicides selected from the
group consisting of an HPPD inhibitor, glyphosate, and glufosinate. In
another embodiment, the locus comprises a transgenic plant that has been
transformed with any combination of nucleic acid molecules described
above, including one or more nucleic acid molecules encoding a mutant
HPPD polypeptide or variant thereof that confers resistance or tolerance
to an herbicide in combination with at least one, at least two, at least
three, or at least four additional nucleic acid molecules encoding
polypeptides that confer desirable traits.

[0242] In one embodiment, the present invention provides transgenic plants
and methods useful for the control of unwanted plant species in crop
fields, wherein the crop plants are made resistant to HPPD chemistry by
transformation to express genes encoding mutant HPPD polypeptides, and
where an HPPD herbicide is applied as an over-the-top application in
amounts capable of killing or impairing the growth of unwanted plant
species (weed species, or, for example, carry-over or "rogue" or
"volunteer" crop plants in a field of desirable crop plants). The
application may be pre- or post emergence of the crop plants or of the
unwanted species, and may be combined with the application of other
herbicides to which the crop is naturally tolerant, or to which it is
resistant via expression of one or more other herbicide resistance
transgenes. See, e.g., U.S. App. Pub. No. 2004/0058427 and PCT App. Pub.
No. WO 98/20144.

[0243] In another embodiment, the invention also relates to a method of
protecting crop plants from herbicidal injury. In the cultivation of crop
plants, especially on a commercial scale, correct crop rotation is
crucially important for yield stability (the achievement of high yields
of good quality over a long period) and for the economic success of an
agronomic business. For example, across large areas of the main
maize-growing regions of the USA (the "central corn belt"), soya is grown
as the subsequent crop to maize in over 75% of cases. Selective weed
control in maize crops is increasingly being carried out using HPPD
inhibitor herbicides. Although that class of herbicides has excellent
suitability for that purpose, it can result in agronomically unacceptable
phytotoxic damage to the crop plants in subsequent crops ("carry-over"
damage). For example, certain soya varieties are sensitive to even very
small residues of such HPPD inhibitor herbicides. Accordingly, the
herbicide resistant or tolerant plants of the invention are also useful
for planting in a locus of any short term carry-over of herbicide from a
previous application (e.g., by planting a transgenic plant of the
invention in the year following application of an herbicide to reduce the
risk of damage from soil residues of the herbicide).

[0244] The following examples are provided by way of illustration, not by
way of limitation.

[0245] The DNA sequence (SEQ ID NO:1) synthesised by GeneArt (Regensburg,
Germany) encoding an HPPD derived from Avena sativa (SEQ ID NO:14) was
cloned into pET24a and expressed in E. coli BL21(DE3) with 50 ng/ml
kanamycin selection as described in PCT App. Pub. No. WO 02/46387.
Overnight cultures grown at 30° C. were used to inoculate
3×1 litre LB in shake flasks at a ratio of 1:100. Cultures were
grown at 37° C., 220 rpm, until an A1cm 600 nm of 0.6-0.8 was
reached, the temperature decreased to 15° C. and induced with 0.1
mM IPTG. Cultures were grown overnight, and cells harvested after 15 min
centrifugation at 10,000 g. Cells were stored at -20° C. until
extraction. A cell pellet from 3 litres of shake flask culture (˜12
g) was thawed in extraction buffer (50 mM Tris, 10 mM sodium ascorbate, 2
mM DTT, 2 mM AEBSF, 10 μM trypsin inhibitor, 1 mM EDTA, pH 7.66) at a
ratio of 1 ml buffer: 1 g cell paste. Extract was passed through the cell
disrupter at 30,000 psi, and centrifuged at 50,000 g for 25 min. at
4° C. Optionally the extract is buffer exchanged down Sepadex G25.
Supernatants were beaded in liquid nitrogen and stored at -80° C.
Levels of HPPD expression were estimated by Western blot analysis and
using purified Avena (1-10 ng) as standard. Extracts were diluted 1:6000
and 1-10 ul were loaded onto 12% SDS PAGE. In addition, expression was
quantified by comparing induced and uninduced SDS PAGE with
COOMASSIE® (Imperial Chemicals Industries, Ltd., London UK) staining
Gels were blotted onto PVDF membrane and Western blots carried out using
rabbit anti-wheat HPPD (1:6600) serum as primary antibody and goat
anti-rabbit FITC-linked antibodies (1:600) as secondary. Detection of
bands was carried out by scanning on a Fluorimager® 595 (GE Healthcare
Ltd, Buckinghamshire UK) and peak quantification was carried out by using
ImageQuant® (GE Healthcare Ltd, Buckinghamshire UK). Plasmid DNA was
reisolated from all transformed strains and the DNA sequence across the
coding region confirmed.

[0246] By Western, the expression level of SEQ ID NO:14 polypeptide
expressed in the E. coli extract was estimated to be about 10-14 mg/ml.
out of a total soluble protein concentration of 33.5 mg/ml.

[0247] The concentration of active HPPD in the extract was also more
accurately estimated by active site titration. For example a range of
volumes of extract (typically 0-20 ul) were added to 50 mM BisTrisPropane
buffer at pH7.0 and at 25° C. containing 25 mM Na ascorbate, 4
μg/ml bovine catalase and 3 nmoles of 14C-labelled compound of
Structure A (1.81 GBq/mmol), in a total final assay volume of 425 μl.

##STR00015##

[0248] The radiolabel protein binding reaction was quenched after 3
minutes by the addition of 100 μl of 1 mM `cold` Structure A. Protein
was exchanged into 50 mM BisTrisPropane buffer at pH 7.0 containing 0.1M
KCl by rapid chromatography down a NAP5 G25 Sephadex column (GE
Healthcare Ltd, Buckinghamshire UK) and 14C bound to protein
fractions measured in Optiphase scintillant using a Tri-Carb 2900TR
scintillation counter (Perkin Elmer, Wellesley, MA). The HPPD binding
site concentration in the extract was calculated from the titration as
described in PCT Patent App. Pub. No. WO 02/46387 and was estimated as
94.9, 78.3, and 82.3 (average 85.2) μM in one extract and 47.2 μM
in another example.

[0249] In an alternate method, the active site titre was calculated on the
basis of an activity-based assay titration carried out by pre-incubating
various ratios of extract and solutions of Structure A in order to
achieve accurate titration of the active site, followed by rapid dilution
into assay solution containing 100-200 μM pHPP for immediate assay by
HPLC/UV quantitation of homogentisate formation after 30-40s (i.e., a
time sufficiently short that inhibitor dissociation and association does
not significantly occur on the timescale of the assay) as described
below.

[0250] The KmHPP and kcat values of the expressed HPPD were estimated
on the basis of assays carried out at 25° C. in solutions of 50 mM
BisTrisPropane buffer at pH 7.0 containing 25 mM Na ascorbate, 4 μg/ml
bovine catalase (Sigma, St. Louis, Mo.), and a range of concentrations
(typically 0.5-10×Km) of 4-hydroxyphenylpyruvate. Typically assays,
in a final volume of 110 μl were started with the addition of enzyme
and accurately stopped after 20 or preferably 10 seconds with whirlimixed
addition of 20 μl 25% perchloric acid. The assay solution was
transferred to Chromacol 03-CVG HPLC vials, sealed and the amount of
homogentisate formed in a 40 μl aliquot determined by injection onto a
reverse phase Aqua C18 5μ 75×4.6 mm HPLC column running 5.5%
acetonitrile 0.1% TFA (Buffer A) at 1.5 ml/min. The column was eluted at
1.5 ml/minute using a 2 minute wash in buffer A, followed by a 2 minute
wash in a 30/70 mixture of buffer A and 100% Acetonitrile, and a further
3.5 minute wash in buffer A. The elution of homogentisate was monitored
by UV at 292 nm and the amount formed in each reaction quantified by
comparison with a standard calibration curve.

[0251] Km and Vmax values were determined (for example FIG. 1) using a non
linear least squares fit using Grafit 4® software (Erithacus Software,
Middlesex, UK). Kcat values were determined by dividing the maximum rate,
Vmax expressed in nmol/second by the number of nmoles of HPPD enzyme
(based on the concentration determined by active-site titration).

[0252] From one set of separate experiments similar to those that produced
the data shown in FIG. 1, on one extract of HPPD SEQ ID NO:14 the Km
value was estimated as 6.17, 4.51, 6.09, 6.13, 4.37, 4.62, 5.41, 5.13 and
6 μM (Km average=5.38 μM). The corresponding kcat values were 4.92,
6.25, 7.08, 6.26, 5.5, 6.77, 6.89, 7.12 and 7.39 s-1 (kcat
average=6.46 s-1). Note that for this calculation and, standardly
herein, Mr was taken to be ˜94 kD and one active-site per dimer was
assumed (i.e., half sites activity as well as inhibitor binding; see
Garcia et al. (2000) Biochemistry, 39:7501-7507; Hawkes
"Hydroxyphenylpyruvate Dioxygenase (HPPD)--The Herbicide Target." In
Modern Crop Protection Compounds. Eds. Kramer and Schirmer. Weinheim,
Germany: Wiley-VCH, 2007. Ch. 4.2, pp. 211-220). If the alternate
assumption of one active site per monomer had been assumed then
calculated kcat values would have been correspondingly halved.

[0253] On rates (governed by an association rate constant, kon) for the
formation of the enzyme:inhibitor complexes, EI and off rates (governed
by a dissociation rate constant, koff) were determined by methods known
in the art and essentially as described in Hawkes et al. (2001) Proc.
Bright. Crop. Prot. Conf. Weeds, 2:563-568 and in PCT Patent App. Pub.
No. WO 02/46387).

[0254] For example, on rates were measured by, at zero time, adding
˜60 pmoles HPPD to 50 mM BisTrisPropane buffer at pH7.0 and at
25° C. containing 25 mM Na ascorbate, 4 μg/ml bovine catalase
(Sigma, St. Louis, Mo.) and an excess (˜300 pmoles) of 14C
inhibitor in a total assay volume of 425 μl and, at various time
points (0-180 s), quenching the radiolabel binding reaction by addition
and rapid mixing of 100 μl `cold` 1 mM structure A. Protein samples
quenched at different times were then exchanged into 50 mM BisTrisPropane
buffer at pH 7.0 containing 0.1M KCl by rapid chromatography down a NAP5
G25 Sephadex column (GE Healthcare Ltd, Buckinghamshire UK) and the
amount of 14C bound to protein fractions quantified in Optiphase
scintillant using a Tri-Carb 2900TR scintillation counter (Perkin Elmer,
Wellesley, Mass.). The data were fit according to the scheme below in
order to derive the value of the apparent second order rate constant, k2,
governing the association rate of enzyme and radiolabelled inhibitor. A
range of enzyme and inhibitor concentrations were used. Optionally, the
rate constant may be derived from similar experiments where enzyme (at
˜0.05-0.2 μM binding sites) and, in this case, unlabelled,
inhibitor (at ˜0.5 to 2 μM) are reacted for a range of short
times (0-60 s) in 50 mM BisTrisPropane buffer at pH7.0 and at 25°
C. containing 25 mM Na ascorbate, 4 μg/ml bovine catalase (Sigma, St.
Louis, Mo.) and then quenched by rapid dilution into assay solution
containing 100-200 μM HPP for immediate assay by HPLC/UV quantitation
of homogentisate formation after 30-40 s (i.e., a time sufficiently short
that inhibitor dissociation and association does not significantly occur
on the timescale of the assay) as described above. Further example
methods are described in PCT Patent App. Pub. No. WO 02/46387.

[0255] Off rates (k1 in the scheme below) were derived from exchange rate
studies where either the test inhibitor, I, or its exchange partner, J
were radiolabelled and the data fit according to the scheme below. As
noted in Hawkes et al. (2001) Proc. Bright. Crop. Prot. Conf. Weeds,
2:563-568, HPPD preparations typically appear to contain 15-30% of a more
rapidly exchanging (weaker binding) fraction of inhibitor binding sites.
This may be a slightly damaged form of the enzyme (it maintains catalytic
activity and may have a higher substrate Km) and, except where off rates
are so fast that fast and slow exchanging fractions are rendered
indistinguishable, off rates always refer to the behaviour of the majorly
slower exchanging fraction that represents 70-85% bulk of the HPPD
inhibitor binding sites present in the extracts tested.

##STR00016##

[0256] Off rates were determined by preincubating, for example, ˜200
pmoles of HPPD binding sites (determined as described above by active
site titration in a 3 min reaction with structure A) in 50 mM
BisTrisPropane buffer at pH 7.0 and at 25° C. containing 25 mM Na
ascorbate, 4 μg/ml bovine catalase (Sigma, St. Louis, Mo.) containing
˜1.0 nmole 14C inhibitor @ 25° C. in a total assay
volume of 1.3 mls. After 30 minutes the exchange reaction was initiated
with addition of 100 μl 1 mM `cold` structure A with thorough mixing,
and, immediately, 150 μl were withdrawn and loaded onto a NAP5 column,
the protein exchanged into 50 mM BisTrisPropane buffer at pH 7.0
containing 0.1M KCl by rapid (<2 min) chromatography down a NAP5 G25
Sephadex column (GE Healthcare Ltd, Buckinghamshire UK) and the amount of
14C bound to protein measured by Optiphase scintillant using a
Tri-Carb 2900TR scintillation counter (Perkin Elmer, Wellesley, Mass.).
Further aliquots were removed and measured in the same way at various
times over minutes or hours as required in order to determine the
exchange kinetics.

[0257] In one variant of the method useful to better distinguish between
off rates that were relatively rapid (e.g., where t 1/2<15 min at
25° C.) the temperature of the experiment was reduced from
25° C. to ice temperature. In this case, off rates were determined
by preincubating ˜200 pmoles HPPD in reaction buffer (50 mM BTP
pH7, 25 mM Na ascorbate, 4 ug/ml bovine catalase, and 10% glycerol)
containing ˜1.0 nmoles 14C inhibitor at 25° C. in a
total assay volume of 1.3 mls. After 30 minutes the reaction vessel was
transferred to ice. After a further 10 minutes at ice temperature the
exchange reaction was initiated by addition of 100 μl 1 mM Structure
A, with thorough mixing, and 150 μl was withdrawn and loaded onto a
NAP5 column in a cold room at ˜5-8° C. in order to quantify
the amount of radiolabel remaining bound to the protein at various time
from the start of exchange at ice temperature.

[0258] Off rates (k1) of HPPD inhibitors that are not available
radiolabelled or that present other measurement problems (for example
high levels of background non-specific protein-binding which can be
measured as radiolabel binding that persists in the presence of high
concentrations of `cold` inhibitor) may be measured indirectly. In this
case the enzyme complex (˜0.1-0.2 μM) is first formed with the
unlabelled inhibitor and then the exchange kinetics derived by chasing it
off with high a concentration of 14C-labelled structure A and
monitoring the rate at which the label becomes bound to protein.
Structure A is a particularly potent inhibitor with known kinetics and in
a 20 fold or more excess will, in equilibrium, >95% occupy the binding
sites in exchange competition with the other inhibitors tested here and
indeed most other inhibitors (those skilled in the art will of course
design the experiment/relative concentrations and fit the data
accordingly). Exemplary methods are also described in PCT Patent App.
Pub. No. WO 02/46387.

[0259] Exemplary on and off rate data (and derived Ki values) were
obtained for the Avena-derived HPPD SEQ ID NO:11 for the following
compounds as follows.

[0264] On rate k2(av)=6.7E+04 s-1 M-1 at 25° C.
(individual experiments yielded k2=6.35E+04, 7.50E+04, 6.2E+04 as
determined by the direct radiochemical method). For mesotrione which has
a relatively fast off rate estimates for on rate based on the
activity-based method were more variable ranging from 4.2E+04 s-1
M-1, 4.9E+04 s-1 M-1 to 7.5 E+04 s-1 M-1 at
25° C.

[0265] Kd was thus estimated from the radiochemical data as 1.16E-08 M
corresponding to a Kd/Km ratio of 0.00217.

[0268] Based on the radiochemical method the estimate of Kd=9.4 E-09M.

[0269] Therefore the estimate of Kd/Km ratio is then=0.0017.

##STR00020##

[0270] Off rate k1=3.96E-05 s-1 at 25° C. as determined using
the direct, radiochemical method (individual measurements of 4.17E-05
s-1 and 3.75E-05 s-1).

[0271] On rate k2=3.20E+04 M-1 s-1 at 25° C. as
determined by the direct radiochemical method. This is in fair agreement
with estimates from the activity based method for on rate of 3.20E+04
M-1 s-1 and 5.7E+04 M-1 s-1.

[0272] Based on the radiochemical methods the estimate of Kd=1.23E-9 M.

[0273] The estimate of Kd/Km ratio=0.00023.

##STR00021##

[0274] Off rate k1=4.17E-05 s-1 at 25° C. as determined by the
indirect, radiochemical method. (individual measurements of 5.50E-05
s-1 and 2.85E-05 s-1).

[0275] On rate k2=1.30E+05 M-1 s-1 at 25° C. as
determined by the direct non-radiochemical method.

[0276] The estimate of Kd=3.21E-10M.

[0277] The estimate of Kd/Km ratio=0.000059.

Example 2

Cloning, Expression and Assay of Further Variants of Avena-Derived HPPDs
SEQ ID NOS:12-20 and Determination of kcat, KmHPP and Ki (kon and
koff) Values Versus Various HPPD Herbicides

[0290] HPPD SEQ ID NO:25 was changed relative to SEQ ID NO:14 by the
substitution of A for L within the sequence motif G(I,V)LVDR (residues
1-6 of SEQ ID NO:30).

[0291] HPPD SEQ ID NO:26 was changed relative to SEQ ID NO:14 by the
substitution of M for L within the sequence motif G(I,V)LVDR (residues
1-6 of SEQ ID NO:30) and by the substitution of M for L within the
sequence motif SGLNS (residues 5-9 of SEQ ID NO:43).

[0292] Values (generally radiochemically determined) of kon (k2), koff
(k1), and Ki (all at 25° C.) were obtained for the HPPDs in the
present example versus the various inhibitor structures as shown in Table
3. The values given for the reference SEQ ID NO:14 in Table 3 are the
average values from a number of experiments as described above. All of
the experiments with the other HPPDs included side by side measurements
with SEQ ID NO:14 as a comparative control. Within experiments, the
ratios of on and off rates relative to this side by side control were
reproducible even where absolute values varied somewhat. Thus the values
given in Table 3 for HPPD SEQ ID NOs:15-26 are normalized versus the
average control values for HPPD SEQ ID NO:14 according to these observed
ratios.

[0293] For example, the off rate of mesotrione (structure B) from HPPD SEQ
ID NO:14 was clearly differentiated from that of SEQ ID NO:24 (see FIGS.
4A-4C) with the goodness of fits being sensitive to small changes in
koff. From these data it can be seen that mesotrione dissociated about
twice as fast from HPPD SEQ ID NO:26 as from HPPD SEQ ID NO:24, and from
HPPD SEQ ID NO:24 about twice as fast as from HPPD SEQ ID NO:14.
Generally the absolute estimates of koff obtained from the fits to the
data were reproducible to within +/-10% and usually better.

[0294] When off rates became relatively fast (t1/2<10 minutes) it was
also useful to make comparative measurements at ice temperature in order
to more accurately confirm the differential between one HPPD and another.
Thus, for example, at ice temperature, mesotrione dissociation from HPPD
SEQ NO:14 was governed by a rate constant, koff, of 1.16E-05 s-1
(much slower than the value of 8.1 E-04 s-1 estimated at 25°
C.) whereas for SEQ ID NOS:22, 24 and 26, the corresponding mesotrione
off rates at ice temperature were 2.17E-05 s-1, 2.25E-05 s-1
and 4.17E-05 s-1; these values being in good proportionate agreement
with those at 25° C. (See Table 3).

[0295] A number of conclusions were derived from the data in Table 3. The
properties of HPPDs SEQ ID NOS:15-17 indicated that certain substitutions
for asparagine(Q) within the amino acid sequence GVQHI provided
significant improvements relative to HPPD SEQ ID NO:14 in tolerance
(slower values of kon and/or faster values of koff), with respect to, for
example, Structures A, B and C.

[0300] Data from HPPD SEQ ID NO:26 indicated that the combination of
certain substitutions for leucine(L) within the amino acid sequence ESGLN
with certain substitutions for leucine (L) within the amino acid sequence
G(I,V)LVDRD provided yet further significant improvements relative to
HPPD SEQ ID NO:14 (and over and above the effect of either single change)
in tolerance (mainly via faster values of koff) with respect to, for
example, Structures B.

[0301] Again, as described for Example 1, kcat and Km values were
determined for a number of the HPPDs of the invention expressed in
extracts and the values are depicted in Table 4.

[0302] A number of the HPPD variants had low Km values similar to HPPD SEQ
ID NO:14 and higher values of Ki/Km with respect to the various HPPD
herbicides and, thus, overexpression in plants expected to provide
enhanced herbicide tolerance to these herbicides. For example, HPPD SEQ
ID NO:24 was twice as resistant to mesotrione as was HPPD SEQ ID NO:14
since it exhibited a Ki/Km ratio of 0.0047 as compared with 0.0021.

[0303] In addition, all of the above sequences as well as libraries of
variants mutated at the same amino positions that showed altered and
enhanced levels of herbicide tolerance are useful to be included in
mutagenesis and shuffling processes in order to generate yet further
shuffled and mutated HPPDs useful as transgenes for conferring herbicide
tolerance. For example, the mutants disclosed in Table 5 are useful for
generating a herbicide tolerant HPPD mutant polypeptide and for inclusion
in recombination reactions to generate further HPPDs.

[0304] As another example, the mutants disclosed in Table 6 are also
useful for generating a herbicide tolerant HPPD mutant polypeptide and
for inclusion in recombination reactions to generate further HPPDs.

[0305] Table 7 summarises data from kinetic studies of a range of mutants
of HPPD SEQ ID NO:14 expressed relative to the control, `none`, meaning
non-mutated HPPD SEQ ID NO:14. Experiments were carried out as described
for Table 4. `Sulc` denotes sulcotrione and `nd` means `no data`. For
V217I, L271I, L271V, V258M and A326R, the relative values of kcat were
estimated from comparisons of the initial rates in cell extracts of
similarly prepared and expressed HPPDs in conventional enzyme activity
assays at pH 7.0, 25° C. and at a substrate concentration of 120
μM HPP. V217I, V258M and A326R, M325L and L358M mutants of SEQ ID
NO:14 are active HPPD enzymes that offer some resistance to sulcotrione,
and may also offer resistance to B. K411T offers significant resistance
to E and especially since the greater than 5× increase in Kd to
this herbicide comprises mainly an improvement in off rate (3.5×)
rather than in on rate. L358M, M325L and K411T all offer improvements
with respect to D. For herbicide tolerance L271I and L271V appear to
offer significant advantages in kcat over unmutated enzyme.

[0306] It will be appreciated that the majority of substitutions to amino
acids within the highly conserved active-site region of HPPD and that lie
within 8° A of the atoms of bound mesotrione (according to
interpretation of published X Ray crystallographic data of the maize and
arabidopsis HPPDs and homology model building to oat HPPD) result in
disabled or only partially functional enzymes. From sequence alignments
of (active) HPPD sequences in the database, about 60 single or double
mutants of SEQ ID NO:14 were selected as amenable to changes in some
residues without loss of enzyme activity (on the basis that they were
changes that represented some of the spread of sequence variation found
amongst natural HPPDs at these positions). These mutants were made,
grown, the HPPDs expressed and extracts prepared and tested for their
catalytic activity and resistance to mesotrione (relative to the control,
unmutated SEQ ID NO:14). Even amongst this privileged set the majority
exhibited significantly impaired catalytic activity and/or were
significantly more sensitive to sulcotrione than the control. Y287F and
I370V were neutral mutations with similar (within 20%) values of kcat and
resistance to sulcotrione as the unmutated enzyme. Amongst a further set
of about 70 mutants encompassing residues as far as 10° A from the
atoms of the bound inhibitors further such neutral mutations (with
respect to SEQ ID NO:14) were G254S, G254A, E416Q, V258M, V258I, V258A,
V258K, S415K, 5415Q, I421L, A326S, L269M, L269F, S420A, T372S, Y172V and
1299M. These further mutations can all be optionally combined with the
resistance conferring mutations to produce catalytically active variants
of HPPD herbicide resistant enzymes of the current invention.

[0307] A further mutant of HPPD SEQ ID NO:14, G408A, exhibited inhibition
kinetics in respect of B and C showing that this mutant was relatively
resistant to inhibition by these compounds. The timecourses of inhibition
were not straightforward and could not be fit to the kinetic model
described above. The experimental method used was similar to that
described above for measuring inhibitor-binding on rates by monitoring
enzyme activity. The time courses of inhibition are depicted in FIGS.
10A-10D. Enzyme at about 75 nM was incubated with inhibitor at 0.15 or
0.6 μM for various times up to 260s and then immediately assayed over
a 150s period following addition of 115 μM HPP (and thus with [S] at
˜30×Km also dramatically slowing any further inhibitor
binding). In the case of the mutant there appeared to be an initial rapid
phase of inhibition which then slowed to leave the enzyme only partly
inhibited. In the case of control enzyme inhibition proceeded to (or was
on course towards) full inhibition. Although note that in the case of
inhibition of the control enzyme by compound B did not quite reach 100%.
The ˜8% residual activity in this case was an artifact of the
method due to the relatively fast off rate of compound B which allowed
some activity to recover during the 150s assay used to monitor the
progress of the reaction between enzyme and inhibitor. This artifact is
negligible with slower dissociating inhibitors such as C. Over the time
of the experiment and at 0.6 μM B, inhibition of mutant G408A appeared
to level off to a residual activity of about 35%. It appeared that this
was not due to an even faster off rate for B from G208A than from the
control enzyme since, at ice temperature, the radiochemically determined
off rate of B from G408A appeared indistinguishable from the rate
observed with the control SEQ ID NO:14 HPPD. Mutant G408 also exhibited a
similar kcat and kcat/Km to SEQ ID NO: 14 HPPD. Whatever the explanation
both B and C appeared to inhibit the G408A mutant HPPD to a lesser extent
than the control enzyme. It is also notable the G408A activity appeared
unstable since the control activity in the absence of inhibitor declined
over the course of the experiment. The addition of inhibitor appeared to
arrest this decline in activity and in further experiments it was
confirmed that mutant G408A activity was unstable in the absence of
inhibitor or substrate but was stabilized by inhibitor and appeared no
less stable than wild type enzyme over extended assay time courses in the
presence of substrate or when partially inhibited by HPPD herbicide.
Thus, despite some instability, mutant G408A is useful alone or in
combination with other mutations to provide useful herbicide tolerance
while herbicide is present in the plant tissues where it is expressed.

[0308] Aside from enzyme kinetic experiments, enhanced resistance to HPPD
herbicides was further demonstrated when the HPPDs of the current
invention were expressed in E. coli and the comparative herbicide
resistances of the various HPPDs assessed visually via the production of
pyomelanin. For example HPPD SEQ ID NO:14 and HPPD SEQ ID NO:24 were
expressed from a pET24 vector in E. coli BL21 cells. Grown without
addition of IPTG there was sufficient expression of HPPD for cultures to
slowly turn brown due to the production of pyomelanin pigment (which
results from auto-oxidation of HPPD-derived homogentisate). Cells were
grown from a 10% starting inoculum into 0.5 ml of L-broth containing 50
μg of kanamycin ml-1 in 45 well plates for 48-96 h at 15°
C. Typically pyomelanin colour in the medium was read (at 430 nm) after
˜72 h. It was noted that addition of 12.5 ppm mesotrione caused
significantly proportionately less inhibition of pyomelanin colour
development in the cells expressing HPPD SEQ ID NO:24 than expressing
HPPD SEQ ID NO:14. FIG. 5 compares the absorbance of the medium obtained
after 72 h in side by side triplicate grows of E. coli expressing HPPD
SEQ ID NOS:14, 18, and 24 all grown in the same plate.

[0309] Cells expressing HPPD SEQ ID NO:24, which exhibited the highest
ratio of Ki/Km, consistently exhibited the least difference in colour
between cells grown with and without 12.5 ppm mesotrione present in the
medium. The same was seen when the mesotrione was replaced with 20 ppm
sulcotrione (data not shown) indicating that SEQ ID NO 24 offers enhanced
tolerance to sulcotrione as well as to mesotrione. Similarly, cells
expressing mutant G408A also exhibited resistance relative to HPPD SEQ ID
NO:14 to sulcotrion according to the pyomelanin assay with 25 ppm
sulcotrione.

[0310] In the present example, mutant HPPD genes derived from Avena HPPD
were the sequences set forth in SEQ ID NOS:1-13 (optimized for tobacco)
or, optionally, are codon optimized according to target crop (e.g.,
soybean) and prepared synthetically and obtained commercially from
GeneArt (Regensburg, Germany). Each sequence is designed to have 5' NdeI
and 3'BamHI sites to facilitate direct cloning. For example, the
sequences set forth in SEQ ID NOS:11, 12, or 13 are cloned into a
suitable binary vector for Agrobacterium-based plant transformation.

[0311] In a particular example genes encoding HPPD SEQ ID NO:14 and HPPD
SEQ ID NO: 24 were cloned into identical expression constructs as
described below and transformed into tobacco.

[0312] As described in PCT Patent App. Pub. No. WO 02/46387, the HPPD
encoding nucleotide sequence is edited by PCR (or initially synthesized)
to include 5' Nco 1 and 3' Kpn 1 sites (and to remove any such internal
sites). This product is then ligated into pMJB1. pMJB1 was a pUC19
derived plasmid which contains the plant operable double enhanced CaMV35S
promoter; a TMV omega enhancer, and the NOS transcription terminator. A
schematic representation of the resulting plasmid is shown in FIG. 2 of
PCT Patent App. Pub. No. WO 98/20144. The expression cassette, comprising
the double enhanced 35S promoter, TMV omega leader, 4-HPPD gene and nos
terminator, is excised using Hind III/Eco R1 (partial Eco R1 digest) and
cloned into similarly digested pBIN 19 and transformed into E. coli TOP
10 competent cells. DNA recovered from the E. coli is used to transform
Agrobacterium tumefaciens LBA4404, and transformed bacteria are selected
on media contain rifampicin and kanamycin. Tobacco tissue is subjected to
Agrobacterium-mediated transformation using methods well described in the
art or as described herein. For example, a master plate of Agrobacterium
tumefaciens containing the HPPD expressing binary vector is used to
inoculate 10 ml LB (L broth) containing 100 mg/1 Rifampicin plus 50 mg/1
Kanamycin using a single bacterial colony. This is incubated overnight at
28° C. shaking at 200 rpm. This entire overnight culture is used
to inoculate a 50 ml volume of LB containing the same antibiotics. Again
this is cultured overnight at 28° C. shaking at 200 rpm. The
Agrobacterium cells are pelleted by centrifuging at 3000 rpm for 15
minutes and then resuspended in MS (Murashige and Skoog) medium
containing 30 g/1 sucrose, pH 5.9 to an OD (600 nM)=0.6. This suspension
is dispensed in 25 ml aliquots into petri dishes.

[0313] Clonally micro-propagated tobacco shoot cultures are used to excise
young (not yet fully expanded) leaves. The mid rib and outer leaf margins
are removed and discarded, and the remaining lamina cut into 1 cm
squares. These are transferred to the Agrobacterium suspension for 20
minutes. Explants are then removed, dabbed on sterile filter paper to
remove excess suspension, then transferred onto solid NBM medium (MS
medium containing 30 g/1 sucrose, 1 mg/1 BAP (benzylaminopurine) and 0.1
mg/1 NAA (napthalene acetic acid) at pH 5.9 and solidified with 8 g/1
Plantagar), with the abaxial surface of each explant in contact with the
medium. Approximately 7 explants are transferred per plate, which are
then sealed and maintained in a lit incubator at 25° C. for a 16
hour photoperiod for 3 days.

[0314] Explants are then transferred onto NBM medium containing 100 mg/1
Kanamycin plus antibiotics to prevent further growth of Agrobacterium
(200 mg/1 timentin with 250 mg/1 carbenicillin). Further subculture onto
this same medium was then performed every 2 weeks.

[0316] Putative transgenic plants that are rooting and showing vigorous
shoot growth on the medium incorporating Kanamycin are analysed by PCR
using primers that amplified a 500 bp fragment within the HPPD transgene.
Evaluation of this same primer set on untransformed tobacco showed
conclusively that these primers would not amplify sequences from the
native tobacco HPPD gene.

[0317] Transformed shoots are divided into 2 or 3 clones and regenerated
from kanamycin resistant callus. Shoots are rooted on MS agar containing
kanamycin. Surviving rooted explants are re-rooted to provide
approximately 70-80 kanamycin resistant and PCR-positive events from each
event.

[0318] Once rooted, plantlets are transferred from agar and potted into
50% peat, 50% John Innes Soil No. 3 with slow-release fertilizer in 3
inch round pots and left regularly watered to establish for 8-12d in the
glass house. Glass house conditions are about 24-27° C. day;
18-21° C. night and approximately a 14 h photoperiod. Humidity is
adjusted to ˜65% and light levels used are up to 2000
μmol/m2 at bench level.

[0319] Two transgenic populations each of about 80 tobacco plants and
comprising, alternatively, an HPPD gene encoding HPPD SEQ ID NO:14 or
HPPD SEQ ID NO:24 within otherwise identical expression cassettes were
thus produced. These two populations were grown on until about the 2-4
leaf stage and then each divided into two subpopulations, one comprising
those plantlets that had emerged rather larger and more advanced from
tissue culture and the other population comprising the smaller plants.
Thus the small sized populations of SEQ ID NO:14 and SEQ ID NO:24
appeared visually similar to comparable each other as did the two
populations of larger sized plants.

[0320] The two smaller populations were each then sprayed with 300 g/ha of
mesotrione and the two larger populations with 500 g/ha. Callisto®
was mixed in water with 0.2-0.25% X-77 surfactant and sprayed from a boom
on a suitable track sprayer moving at 2 mph with the nozzle about 2
inches from the plant tops. Spray volume was 2001/ha.

[0321] Plants were assessed for damage and scored at 13 days after
treatment (DAT). All four populations appeared highly resistant to the
herbicide treatments but the SEQ ID NO:24 HPPD expressing populations
more so than the control SEQ ID NO:14 populations. From the two
larger-sized populations sprayed with 500 g/ha only 4 of 38 (10%)
morphologically normal PCR positive plants (one emerged chimeric)
expressing SEQ ID NO:24 exhibited symptoms of herbicide damage whereas 9
out of a total of 33 (27%) of SEQ ID NO:14 expressing plants exhibited
damage. There was little damage to see on the two smaller-sized
populations sprayed with 300 g/ha mesotrione; here 2 of 28 SEQ ID NO:24
expressing plants exhibited visible herbicide damage as compared with 4
of 26 SEQ ID NO:14 expressing plants.

[0322] Plants of events showing the least damage are grown to flowering,
then bagged and allowed to self. The seed from selected events are
collected and sown again in pots, and tested again for herbicide
resistance in a spray test for resistance to HPPD herbicide (for example
mesotrione). Single copy events amongst the T1 plant lines are identified
by their 3:1 segregation ratio (with respect to kanamycin and/or
herbicide) and by quantitative RT-PCR. Seed from the thus selected T1
tobacco (var. Samsun) lines are sown in 3 inch diameter pots containing
50% peat and 50% John Innes Soil No. 3.

Example 4

Construction of Soybean Transformation Vectors

[0323] Binary vectors for dicot (soybean) transformation were constructed
with a promoter, such as a synthetic promoter containing a CaMV 35S and
an FMV transcriptional enhancer and a synthetic TATA box driving the
expression of an HPPD coding sequence, such as SEQ ID NO:24, followed by
Nos gene 3' terminator. The HPPD gene was codon-optimized for soybean
expression based upon the predicted amino acid sequence of the HPPD gene
coding region. In the case that HPPD itself was not used as the
selectable marker, Agrobacterium binary transformation vectors containing
an HPPD expression cassette were constructed by adding a transformation
selectable marker gene. For example, binary transformation vector 17146
(SEQ ID NO:33) contains an expression cassette for HPPD variant (SEQ ID
NO:24) linked with two PAT gene cassettes (one with the 35S promoter and
one with the CMP promoter, and both PAT genes are followed by the nos
terminator) for glufosinate based selection during the transformation
process. Another binary transformation vector (17147) (SEQ ID NO:34)
contains the HPPD variant (SEQ ID NO:24) expression cassette and also an
EPSPS selectable marker cassette. Vector 17147 was transformed into
soybean and transgenic plants were obtained using glyphosate selection
after Agrobacterium-mediated transformation of immature seed targets.
Similarly, binary vector 15764, (SEQ ID NO:35) was constructed to
comprise expression cassettes to express HPPD (SEQ ID NO:14) along with a
bar selectable marker gene and binary vector 17149 (SEQ ID NO:36) was
constructed to comprise an expression cassette expressing HPPD variant
(SEQ ID NO:26) along with two PAT gene cassettes. In all cases the DNA
sequences encoding the HPPD genes were codon-optimized for expression in
soybean.

[0324] The Binary Vectors described above were constructed using a
combination of methods well known to those skilled in the art such as
overlap PCR, DNA synthesis, restriction fragment sub-cloning and
ligation. Their unique structures are made explicit in FIGS. 6 (vector
17146), 7 (vector 17147), 8 (vector 15764), and 9 (vector 17149), and in
the sequence listings (SEQ ID NOS:33-36). Additional information
regarding the vectors shown in FIGS. 6-9 are provided below.

[0325] The abbreviations used in FIG. 6 (vector 17146) are defined as
follows:

[0591] Soybean plant material can be suitably transformed and fertile
plants regenerated by many methods which are well known to one of skill
in the art. For example, fertile morphologically normal transgenic
soybean plants may be obtained by: 1) production of somatic embryogenic
tissue from, e.g., immature cotyledon, hypocotyl or other suitable
tissue; 2) transformation by particle bombardment or infection with
Agrobacterium; and 3) regeneration of plants. In one example, as
described in U.S. Pat. No. 5,024,944, cotyledon tissue is excised from
immature embryos of soybean, preferably with the embryonic axis removed,
and cultured on hormone-containing medium so as to form somatic
embryogenic plant material. This material is transformed using, for
example, direct DNA methods, DNA coated microprojectile bombardment or
infection with Agrobacterium, cultured on a suitable selection medium and
regenerated, optionally also in the continued presence of selecting
agent, into fertile transgenic soybean plants. Selection agents may be
antibiotics such as kanamycin, hygromycin, or herbicides such as
phosphonothricin or glyphosate or, alternatively, selection may be based
upon expression of a visualisable marker gene such as GUS. Alternatively,
target tissues for transformation comprise meristematic rather than
somaclonal embryogenic tissue or, optionally, is flower or flower-forming
tissue. Other examples of soybean transforamtions can be found, e.g. by
physical DNA delivery method, such as particle bombardment (Finer and
McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; McCabe et al.
(1988) Bio/technology 6:923-926), whisker (Khalafalla et al. (2006)
African J. of Biotechnology 5:1594-1599), aerosol bean injection (U.S.
Pat. No. 7,001,754), or by Agrobacterium-mediated delivery methods
(Hinchee et al. (1988) Bio/Technology 6:915-922; U.S. Pat. No. 7,002,058;
U.S. Patent App. Pub. No. 20040034889; U.S. Patent App. Pub. No.
20080229447; Paz et al. (2006) Plant Cell Report 25:206-213). The HPPD
gene can also be delivered into organelle such as plastid to confer
increased herbicide resistance (U.S. Patent App. Pub. No. 20070039075).

[0592] Soybean transgenic plants can be generated with the above described
binary vectors (Example 4) containing HPPD gene variants with different
transformation methods. Optionally, the HPPD gene can provide the means
of selection and identification of transgenic tissue. For example, a
vector was used to transform immature seed targets as described (U.S.
Patent App. Pub. No. 20080229447) to generate transgenic HPPD soybean
plants directly using HPPD inhibitor, such as mesotrione, as selection
agent. Optionally, HPPD genes can be present in the polynucleotide
alongside other sequences which provide additional means of
selection/identification of transformed tissue including, for example,
the known genes which provide resistance to kanamycin, hygromycin,
phosphinothricin, butafenacil, or glyphosate. For example, different
binary vectors containing PAT or EPSPS selectable marker genes as
described in Example 4 were transformed into immature soybean seed target
to generate HPPD herbicide tolerant plants using Agrobacterium-mediated
transformation and glufosinate or glyphosate selection as described (U.S.
Patent App. Pub. No. 20080229447).

[0593] Alternatively selectable marker sequences may be present on
separate polynucleotides and a process of, for example, co-transformation
and co-selection is used. Alternatively, rather than a selectable marker
gene, a scorable marker gene such as GUS may be used to identify
transformed tissue.

[0594] An Agrobacterium-based method for soybean transformation can be
used to generate transgenic plants using glufosinate, glyphosate or HPPD
inhibitor mesotrione as selection agent using immature soybean seeds as
described (U.S. Patent App. Pub. No. 20080229447).

[0596] After plants became established in the soil and new growth appeared
(˜1-2 weeks), plants were sampled and tested for the presence of
desired transgene by TAQMAN® analysis using appropriate probes for
the HPPD genes, or promoters (for example prCMP). Positive plants were
transplanted into 4'' square pots containing Fafard #3 soil. Sierra
17-6-12 slow release fertilizer was incorporated into the soil at the
recommended rate. The plants were then relocated into a standard
greenhouse to acclimatize (˜1 week). The environmental conditions
were: 27° C. day; 21° C. night; 14 hr photoperiod (with
supplemental light); ambient humidity. After acclimatizing (˜1
week), the plants were sampled and tested in detail for the presence and
copy number of inserted transgenes. Transgenic soybean plants were grown
to maturity for T1 seed production. T1 plants were grown up, and after
TAQMAN® analysis, homozygous plants were grown for seed production.
Transgenic seeds and progeny plants were used to further evaluate their
herbicide tolerance performance and molecular characteristics.

[0597] Homozygous soybean plants from 2 events made with vector 15764
(FIG. 8) and multiple events made with vector 17147 (FIG. 7) expressing
SEQ ID NO:14 and SEQ ID NO:24, respectively, from identical HPPD
expression cassettes were grown and tested for tolerance to a range of
HPPD herbicide. Table 8 summarises the results of these tests from plants
sprayed at the V2 growth stage. Each data point represents the average
damage score from n=7 replicates.

[0598] Event 1 was most tolerant to mesotrione, and event 2 was the second
most tolerant 15764 event selected from a population of about ninety.
These events were used to compare the performance of five 17147 events.
Four of these, SF, S8, S7 and S3 had not been preselected for tolerance
level (other than to confirm resistance, non-chimerical nature and the
presence of the gene) while the remaining event, TO, had been preselected
as the most resistant out of five 17147 events in a preliminary field
test.

[0600] It is striking that, from such a small pool of 17147 events all
five tested provided tolerance to mesotrione and to isoxaflutole
equivalent to one of the best 15764 events, event 2, and indeed that two
of them, T0 and S7 exceed the performance of the most tolerant 15764
event, event 1, that was selected from many.

[0601] The in vitro data, and in particular the off rate data, show that
SEQ ID NO:24 is 2 and 2.3 fold superior to SEQ ID NO:14 in respect of B
and IFT but neutral in respect of C and E. In accord with this is the
fact that the SEQ ID NO:24 HPPD expressing plants displayed a similarly
altered pattern of herbicide tolerance. Thus, for example, events SF and
S8 exhibits similar or better tolerance to both IFT and B than does 6W
but, unlike 6W, essentially no tolerance to either compound E or C.
Similarly, the only 17147 events, T0 and S7, to exhibit tolerance to E
and C that is close to that of event 4R also exhibit superior tolerance
than 4R to B and IFT. The in vitro data have predictive value in planta
and SEQ ID NO:24 provides improved tolerance to mesotrione and IFT but
not, for example, to tembotrione.

[0602] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the art to
which this invention pertains. All publications and patent applications
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

[0603] Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, certain changes and modifications may be practiced within
the scope of the appended claims.